GENETICS

Molecular Phylogeny and Biogeography of Heterotermes(Isoptera: Rhinotermitidae) in the West Indies

ALLEN L. SZALANSKI,1 RUDOLF H. SCHEFFRAHN,2 JAMES W. AUSTIN,1 JAN KRECEK,2

AND NAN-YAO SU2

Ann. Entomol. Soc. Am. 97(3): 556Ð566 (2004)

ABSTRACT A phylogenetic analysis of Heterotermes from 63 locations in the West Indies andassociated mainland is presented based on the DNA sequence of a portion of the mitochondrial 16SrRNA gene. Maximum parsimony analysis of the aligned Heterotermes species and the outgroup taxaresulted in a consensus tree with Þve distinct West Indian groups. Morphological characters andgeographical distribution supported the resulting clades, which were given the following taxonomicassignments: Heterotermes cardini (Bahamas and Cuba); Heterotermes sp. (Bonaire, FL; Grand Cay-man; Grand Turk; Jamaica; and St. Barthelemy), Heterotermes convexinotatus (Antigua, Barbados,Curacao, Dominican Republic, Martinique, Puerto Rico, St. Kitts, St. Maarten, Venezuela, and VirginIsland), and Heterotermes tenuis (Lesser Antilles except Barbados and Martinique, Ecuador, andSuriname). The Florida Heterotermes sp. is a known nonendemic population, whereas those onBarbados, Grand Cayman, Grand Turk, Martinique, St. Barthelemy, and elsewhere may harbor orconsist solely of nonendemic populations.

KEY WORDS invasive species, DNA sequence, genetic variation, molecular diagnostics, termite

THE TERMITE GENUS Heterotermes Froggatt has centersof abundance and diversity in the warm Neotropics,Australia, the Indian subcontinent, and the Arabianpeninsula (Emerson 1971). Heterotermes distributionis mutually exclusive with that of Reticulitermes, withthe latter occurring in more temperate Holarcticzones as well as from southern China to Vietnam. Inthe Neotropics, Heterotermes spp. are widespread ter-mites on the mainland (Constantino 1998, 2000) andinhabit most islands of theWest Indies (Scheffrahn etal. 1994, and references therein). Three species ofNeotropical Heterotermes are reported from theWestIndies (Snyder 1924, 1949, Constantino 1998):Hetero-termes cardini(Snyder)described fromAndros Island,Bahamas; Heterotermes convexinotatus (Snyder) de-scribed from Panama, and Heterotermes tenuis (Ha-gen) described from a divergent series from Hispani-ola, Colombia, and Brazil. Additionally, there are sixother described South American Heterotermes spp.and Heterotermes aureus (Snyder) from the southernNearctic for a total of 10 New World species.Nonrobust morphological characters and lack of

material, especially the imago caste, has made thespecies level identiÞcation of West Indian Heterot-ermes difÞcult (Adamson 1938, Light 1933, Scheffrahnet al. 1994). In the Þrst diagnosis of New World Het-erotermes, Snyder (1924) placed doubt on the validity

of the three West Indian species in a preamble to hisdescriptions of H. cardini and H. convexinotatus. Sny-der (1956) listed H. cardini, H. convexinotatus, and H.tenuis from Cuba and Hispaniola; H. convexinotatusand H. tenuis from Puerto Rico and Jamaica; and H.cardini and H. tenuis from Bahamas, suggesting un-certainty at species level identiÞcation rather thanwidely sympatric or disjuncted West Indian distribu-tions.Recent studies of termite phylogeny have focused

on higher level determinations by using both mor-phological (Donovan et al. 2000) and molecular (Ka-mbhampati et al. 1996) characters. Some studies havefocused on speciÞc economic genera within the Rhi-notermitidae, including interspeciÞc genetic variationofReticulitermes (Jenkins et al. 1998, 1999; Clement etal. 2001; Marini and Mantovani 2002; Austin et al.2004), and intraspeciÞcvariationofCoptotermes(Hus-senederandGrace2001, Jenkinset al. 2002).However,no studies have focused on intra- or interspeciÞc ge-netic variation of the third major rhinotermitid pestgenus Heterotermes.Information on how genetic variation is partitioned

within populations and among termite species can beuseful for the development of molecular diagnosticsfor identifying species (Szalanski et al. 2003). The 16SrRNA region of the mitochondrial DNA (mtDNA)genome has proved useful for studying the phyloge-netic relationshipof termite families (Kambhampati etal. 1996, Thompson et al. 2000), as well as variationwithin and amongReticulitermes species (Austin et al.2002, 2004; Szalanski et al. 2003).

1 Department of Entomology, University of Arkansas, Insect Ge-netics Research Laboratory, Fayetteville, AR 72701.

2 Department of Entomology and Nematology, University of Flor-ida-Ft. Lauderdale Research and Education Center, 3205 CollegeAve., Ft. Lauderdale, FL 33314.

0013-8746/04/0556Ð0566$04.00/0 � 2004 Entomological Society of America

The purpose of the current study is to report on thedistribution of Heterotermes in the West Indies, com-pare base pair sequences from themtDNA16S gene ofbroadly collected specimens, compare their morpho-logical characters, and relate these to their distribu-tion.

Materials and Methods

Samples of Heterotermes spp. were collected fromthe West Indies and North and South America andpreserved in 85% ethanol (Table 1). Measurements ofimagoes and soldiers from the same and similar local-ities used in genetic studies below were taken with astereomicroscope Þtted with an ocular micrometer.Measurements of winged imagoes, when available,included total length, fore wing length, head width,pronotum width, and fore wing width (Table 2). Sol-dier measurements included head length, head width,and pronotum width. These measurements were se-lectedbecause theybest convey the relative sizeof thespecimens. Voucher specimens, preserved in 85% eth-anol, are maintained at the Arthropod Museum, De-partment of Entomology, University of Arkansas, Fay-etteville, AR, and at the University of Florida FortLauderdale Research and Education Center.ForDNAextraction, specimenswere allowed todry

on Þlter paper, and DNA was extracted from individ-ual worker or soldier heads by using the PuregeneDNA isolation kit D-5000A (Gentra, Minneapolis,MN). Extracted DNA was resuspended in 50 �l ofTris/EDTA and stored at �20�C. Polymerase chainreaction (PCR) was conducted using the primers LR-J-13007 (5�-TTACGCTGTTATCCCTAA-3�) (Kamb-hampati and Smith 1995) and LR-N-13398 (5�-CGC-CTGTTTATCAAAAACAT-3�) (Simon et al. 1994).These PCR primers amplify an �428-base pair (bp)region of the mtDNA 16S rRNA gene. PCR reactionswere conducted using 1 �l of the extracted DNA perSzalanski et al. (2000), with a proÞle consisting of 35cycles of 94�C for 45 s, 46�C for 45 s, and 72�C for 45 s.AmpliÞed DNA from individual termites was puriÞedand concentrated using Microcon-PCR Þlter units(MilliporeCorp., Bedford,MA). Sampleswere sent toUniversity of Arkansas for Medical Sciences DNA Se-quencing Core Facility (Little Rock, AR) for directsequencing in both directions by using an ABI Prism377 DNA sequencer (Foster City, CA). GenBank ac-cession numbers for the Heterotermes termites sub-jected to DNA sequencing in this study are presentedinTable 1. Consensus sequences for each samplewereobtained using Bioedit 5.09 (Hall 1999), and se-quenceswere aligned usingCLUSTALW(Thompsonet al. 1994). Mitochondrial DNA haplotypes werealignedusingMacClade version 4 (SinauerAssociates,Sunderland, MA).The distancematrix option of PAUP* 4.0b10 (Swof-

ford 2001) was used to calculate genetic distancesaccording to the Kimura 2-parameter model (Kimura1980) of sequence evolution. Mitochondrial DNA se-quences of H. aureus from Santa Rita, AZ; H. sp. fromTacoa, Venezuela, and Xunatunich ruins, Belize, and

Chankah Vera Cruz, Mexico (Table 1) were added tothe West Indian Heterotermes data set for phyloge-netic analysis. Nasutitermes costalis (Holmgren)(Isoptera: Termitidae) (GenBank AY486438) andKalotermes flavicollis F. (Isoptera: Kalotermitidae)(GenBank AY486437) were used as the outgroup taxafor the Heterotermes data set. These two taxa wereselected based on their molecular phylogenetic rela-tionship toRhinotermitidae(Kambhampati et al. 1996,Austin et al. 2004).DNAsequenceswere alignedusingthe PILEUP program in GCG (Genetics ComputerGroup, Madison, WI) and adjusted manually. Maxi-mum likelihood and unweighted parsimony analysison the alignments were conducted using PAUP*4.0b10 (Swofford 2001). Gaps were treated as missingdata. The reliability of trees was tested with a boot-strap test (Felsenstein 1985). Parsimony bootstrapanalysis included 1000 resamplings by using theBranch andBound algorithmofPAUP*. Formaximumlikelihood analysis, the default likelihood parametersettings were used (HKY85 six-parameter model ofnucleotide substitution, empirical base frequencies)with theexceptionof the transition/transversion ratio,which was set to 1.399331:1). These parameters wereused to carry out a heuristic search using PAUP*, byusing either the single most parsimonious tree as thestarting tree, or stepwise addition.

Results

Heterotermes records for the West Indies were lastreviewed by Scheffrahn et al. (1994). Since then,ScheffrahnandSu(1995) reportedanewnonendemicestablishment of a Heterotermes sp. in Miami, FL, andScheffrahn et al. (2003a) recordedHeterotermes sp. onVieques and Culebra (Puerto Rico) and Buck and St.John (U.S. Virgin Islands) Scheffrahn et al. (2003b)added Monos, Huevos, and Little Tobago to the Het-erotermes localities of Trinidad and Tobago. Newrecords forHeterotermes reportedherein includeAck-lins, Anguilla, Aruba, Barreterre (Exuma), Bonaire,Cat, Crooked, Curacao, Eleuthera, Great Exuma,Great Inagua, Grenada, Isla de Pinos (Cuba), LittleExuma, Long Island, Mangrove Cay, Marie Galant,Nevis, North Bimini, North Cat Cay, Paradise, RumCay, San Salvador, South Andros, South Bimini, St.Barthelemy, St. Eustatius, St. Kitts, St. Lucia, and St.Maarten.Extensive collecting yielded no Heterotermes sp. on

Abaco, Grand Bahama, Barbuda, Cayman Brac, EastCaicos, Little Cayman, La Desirade, Chacachacare(Trinidad), Domininca, Mayaguana, Middle Caicos,North Caicos, Saba, Saona (Dominican Republic),South Caicos, and West Caicos. A record of Heterot-ermes on Grand Bahama (Scheffrahn et al. 1994) is inerror as this genus is displaced by Reticulitermes fla-vipes (Kollar) on that island as well as on neighboringAbaco (Scheffrahn et al. 1999). Heterotermes was re-corded on Dominica by Collins (Scheffrahn et al.1994) and by Snyder (1956), but vouchers are notavailable and we did not collect it during a survey ofthe island in 1995. A single Heterotermes soldier, not

May 2004 SZALANSKI ET AL.: Heterotermes GENETICS 557

used in this study, was collected on Providenciales(R.H.S., unpublished data).DNA sequencing of the amplicon revealed that it

averaged 428 bp. The average base frequencies were

A� 0.25, C� 0.12,G� 0.22, andT� 0.41. The alignedDNA data matrix, including the outgroup taxa (avail-able atTreeBASE, http://www.treebase.org, study ac-cession number SN1582) resulted in a total of 450

Table 1. Heterotermes collection data, haplotypes, and GenBank accessions

Species Island or location Island or country Haplotype GenBank

H. tenius Barique Grenada A AY380238H. tenius Garview St. Vincent B AY380239H. tenius St. Lucia St. Lucia B AY380240H. tenius Lower Manzanilla Trinidad B AY380241H. tenius NW Roxborough Tobago B AY380242H. tenius Huevos Island Trinidad/Tobago B AY380243H. tenius Little Tobago Trinidad/Tobago B AY380244H. tenius Swamp W Pointe-a-Pitre airport Guadeloupe C AY380245H. tenius Rain Forest Guadeloupe C AY380246

Trail AboveSainte-Rose

H. tenius Anse la Frais Marie Galant C AY380247H. tenius Cerro Blanco Ecuador AY380248H. sp. “Northside” Grand Cayman D AY380249H. sp. Playa Chikitu Bonaire D AY380250H. sp. Hellshire Point Jamaica E AY380251H. sp. Mahoe Bay Jamaica E AY380252H. sp. Negril Jamaica E AY380253H. sp. Dade Co., FL US F AY380254H. sp. N. Gustavia St. Barthelemy G AY380255H. cardini Great Exuma Bahamas H AY380256H. cardini South Andros Bahamas I AY380257H. cardini Long Island Bahamas I AY380258H. cardini Great Exuma Bahamas I AY380259H. cardini Cat Island Bahamas I AY380260H. cardini Crooked Island Bahamas I AY380261H. cardini Cat Island Bahamas I AY380262H. cardini North Andros Bahamas I AY380263H. cardini North Cat Cay Bahamas I AY380264H. cardini Eleuthera Bahamas I AY380265H. cardini Acklins Island Bahamas I AY380266H. cardini Barreterre (Exuma) Bahamas I AY380267H. cardini Little Exuma Bahamas I AY380268H. cardini North Bimini Bahamas I AY380269H. cardini South Bimini Bahamas I AY380270H. cardini New Providence Bahamas I AY380271H. cardini Guantanamo Bay Cuba I AY380272H. cardini San Salvador Bahamas J AY380273H. cardini Rum Cay Bahamas J AY380274H. cardini Mangrove Cay Bahamas K AY380275H. sp. Grand Turk Turks & Caicos L AY380276H. convexinotatus E. Pares Antigua M AY380277H. convexinotatus Basses Terres St. Maarten N AY380278H. convexinotatus Bani Dominican Rep. O AY380279H. convexinotatus St. Kitts St. Kitts-Nevis P AY380280H. convexinotatus Zeelandia Beach St. Eustatius Q AY380281H. convexinotatus JoeÕs River Forest Barbados Q AY380282H. convexinotatus Santo Domingo Dominican Rep. Q AY380283H. convexinotatus Bellefontaine Martinique Q AY380284H. convexinotatus Palm Beach Aruba Q AY380285H. convexinotatus Santa Barbara Curacao Q AY380286H. convexinotatus Hwy 200 S. of airport Vieques Q AY380287H. convexinotatus 1km W Dept. Nat. Res. Culebra Q AY380288H. convexinotatus Hwy 60 nr Great Pond St. Croix Q AY380289H. convexinotatus Cowpet Bay St. Thomas Q AY380290H. convexinotatus Buck Island US Virgin Is Q AY380291H. convexinotatus Monos Island Trinidad/Tobago Q AY380292H. convexinotatus Indian Castle Estates Nevis R AY380293H. convexinotatus Danish Road St. John R AY380294H. convexinotatus Humacao Puerto Rico S AY380295H. convexinotatus West End Village Anguilla T AY380296H. cardini Great Inagua Bahamas U AY380297H. convexinotatus Tacoa Venezuela AY380298H. aureus Santa Rita, AZ US AY380299H. sp Vera Cruz Mexico AY380300H. sp. Xunatunich ruins Belize AY380301

558 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 97, no. 3

characters. Of these characters, 141 (31%) were vari-able and 64 (14%) were phylogenetically informative.This data set had only one most parsimonious tree

(Fig. 1) (length � 225, CI � 0.764), as documentedusing the Branch and Bound search algorithm ofPAUP*. Bootstrap analysis of the alignedHeterotermesspecies and the outgroup taxa resulted in a consensustree with several distinct branches. These distinctclades included H. cardini, from the Bahamas andCuba; Heterotermes sp. from Bonaire, FL; Grand Cay-man; Grand Turk Jamaica; and St. Barthelemy; H.convexinotatus from Antigua, St. Maarten, Barbados,Curacao, the Dominican Republic, Martinique, theU.S. Virgin Islands, and Venezuela; H. tenuis fromGrenada, Guadeloupe, St. Lucia, St. Vincent, TrinidadandTobago, andEcuador, andH. aureus fromArizona;andHeterotermes sp. two fromMexico andBelize (Fig.1).Among the 63 West Indian and other Heterotermes

samples subjected to DNA sequencing, a total of 84nucleotides (20%) were variable and segregated theWest Indian Heterotermes into 21 distinct haplotypes(Table 1; Fig. 2). Pairwise Tajima-Nei distances (Ta-jima and Nei 1984) among the Heterotermes haplo-types ranged from 0.2% between haplotypes E (Ja-maica) and F (Dade County, Florida) to 10.2%between haplotypes B (St. Vincent) and P (St. Kitts)(Table 3). The nonendemic Heterotermes populationin Miami, FL, was most similar to the sample fromJamaica in terms of genetic divergence, differing byonly one nucleotide site.Phylogenetic analysis of the 21West IndianHetero-

termes haplotypes was conducted usingN. costalis andK. flavicollis as the outgroup taxa. This data set hadonly one most parsimonious tree (Fig. 3) (length �184, CI� 0.853), as documented using the Branch andBound search algorithm of PAUP*. Bootstrap analysisof the aligned Heterotermes species and the outgrouptaxa resulted in a consensus tree with several distinctgroups. Group I consisted of haplotypes A, B, and C(Grenada, St. Vincent, and Guadeloupe); group IIwith haplotypes D, E, and F (Jamaica, Cayman Island,and Florida); group III with haplotypes G and L(Grand Turk and St. Barthelemy); group IVwith hap-lotypes H, I, J, K, and U (Bahamas and Cuba); andgroup V with haplotypes M, N, O, P, Q, R, S, and T

(Antigua, St. Maarten, Dominican Republic, St. Kitts,Puerto Rico; Figs. 3 and 5). Regardless of whether thestarting tree was the most parsimonious tree or wasobtained via stepwise addition, the maximum likeli-hood search found only one tree (Fig. 4). The maxi-mum likelihood tree differed from the maximum par-simony tree in that haplotype D did not form a sistergroup with haplotypes E and F.Measurements of Heterotermes imagos and soldiers

(Table 2) generally reßect groupings derived bymax-imum parsimony analysis by using PAUP* (Fig. 2);thus, supporting species designations. Group I, repre-sents specimens that aremorphologically closest toH.tenuis as suggested by the larger sizes of all castes(soldier and imagoheadwidth 0.93Ð1.07 and 1.00Ð1.03mm, respectively; Emerson 1925), a dimorphic soldierhaving tergiteswith bristles and one-third length hairsin interior (Constantino 2000), and the emarginatedposterior edge of themeso- andmetanota of the imago(Snyder 1924). Constantino (2000) reports that H.tenuis is widespread and common in mainland SouthAmerica. Therefore, it is not surprising that samplesfrom Ecuador, Suriname, and Trinidad belong to thisspecies likely constitute over-water dispersal sourcesforH. tenuis in the Lesser Antilles. Group IV, a smallerspecies from the Bahamas and Cuba (soldier andimago head width 0.78Ð0.85 and 0.78Ð0.82 mm, re-spectively; Snyder 1924) having convex posterior bor-ders on the imago meso- and metanota, is morpho-logically consistent with H. cardini. Groups V and VIrepresent specimens from both coastal Venezuela,Hispaniola, PuertoRico, and theirneighboring islands.The group V and VI specimens are smaller than H.tenuis (soldier headwidth 0.79Ð0.84mm,Constantino2000; alate head width 0.8 mm, Snyder 1924), andimagos have convex posterior borders on their meso-and metanota and tergites with posterior bristles andmicroscopic hairs in interior, indicating that theyare conspeciÞc with H. convexinotatus. Constantino(2000) reports numerous localities of H. convexinota-tus from northern Venezuela, whereas Snyder (1956)lists West Indian localities from Barbados, Hispaniola,Jamaica, Puerto Rico, St. Croix, and St. Thomas. Theclade comprising groups II and III, are morphologi-cally nearest to H. cardini; however, these may con-stitute a separate species or subspecies based on their

Table 2. Measurements (means in millimeters of West Indian Heterotermes soldiers and winged imagos as sorted by haplotypegroupings (see Figs. 2, 3, and 5)

Haplotypegroup

Speciesdesignation

SOLDIER IMAGO

nHeadlength

Headwidth

Pronotumwidth

nTotallength

Fore winglength

Headwidth

Pronotumwidth

Fore wingwidth

I H. tenuis 11 2.77a 1.01a 0.78a 8 10.95a 8.73a 0.97a 0.80a 2.01aV H. convexinotatus 17 2.37b 0.87b 0.69b 8 9.87b 7.80b 0.85b 0.75b 1.84aVI H. convexinotatus 4 2.31bc 0.85bc 0.68bc 1 9.59b 7.43b 0.87b 0.75b 1.88aII H. sp. 6 2.24bc 0.83bc 0.63cd 4 10.09b 8.14ab 0.84b 0.72b 1.93aIII H. sp. 2 2.15c 0.83bc 0.63cdIV H. cardini 20 2.12c 0.80c 0.61d 5 9.80b 7.72b 0.84b 0.68c 1.71a

Only large soldiers were measured for dimorphic H. tenuis (group I).a Means of four replicates, 30 nymphs per replicate. Means within a column followed by the same letter are not signiÞcantly different

(StudentÐNewmanÐKeuls test, P � 0.5).

May 2004 SZALANSKI ET AL.: Heterotermes GENETICS 559

genetic dissimilarities. Group II is confounding geo-graphically due to its alignment with a sample fromdistant Bonaire. This species may readily infest boatsas observed in 1995 when a mature colony of Hetero-termes was discovered on a yacht docked in Grand

Caymanwhere it was presumed to have been infested(R.H.S., unpublished). Haplotype group III may alsobe more prone to human transport as suggested bydissociated populations on Grand Turk and St. Bar-thelemy.

Fig. 1. Single most parsimonious tree during a branch and bound search by using PAUP* (Swofford 2001). Bootstrapvalues for 1000 replicates are listed above the branches supported at �50%.

560 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 97, no. 3

Fig. 2. Nucleotide diversity among 21 West Indian Heterotermes haplotypes.

May 2004 SZALANSKI ET AL.: Heterotermes GENETICS 561

Discussion

In this study, a phylogenetic analysis of Heterot-ermes from 63 locations from the West Indies andNorth and South America, based on the DNA se-quence of a portion of the mitochondrial 16S rRNAgene, is presented. Most of the inferred relationships

had strong quantitative support as indicated by boot-strap analysis.Literature on the biogeography ofWest Indianßora

and fauna is growing, andevidencepoints towardbothover-water dispersal and vicariance asmechanisms forpresent day distributions (Woods and Sergile 2001).

Table 3. Tajima-Nei pairwise genetic distances among Heterotermes haplotypes (H)

H A B C D E F G H I J K L M N O P Q R S T U

AB 0.005C 0.007 0.007D 0.095 0.100 0.097E 0.097 0.102 0.099 0.002F 0.100 0.104 0.102 0.005 0.002G 0.092 0.097 0.094 0.007 0.005 0.007H 0.095 0.100 0.097 0.014 0.016 0.019 0.016I 0.095 0.100 0.097 0.012 0.014 0.016 0.014 0.002J 0.097 0.102 0.099 0.014 0.016 0.019 0.016 0.005 0.002K 0.095 0.100 0.097 0.012 0.014 0.016 0.014 0.007 0.005 0.007L 0.099 0.104 0.101 0.012 0.009 0.012 0.005 0.021 0.019 0.021 0.019M 0.094 0.097 0.092 0.061 0.064 0.066 0.059 0.064 0.064 0.066 0.068 0.063N 0.090 0.092 0.089 0.054 0.056 0.059 0.052 0.056 0.057 0.059 0.061 0.056 0.007O 0.097 0.099 0.094 0.063 0.066 0.068 0.061 0.066 0.066 0.068 0.070 0.068 0.023 0.021P 0.099 0.102 0.097 0.068 0.070 0.073 0.066 0.070 0.071 0.073 0.075 0.070 0.012 0.014 0.012Q 0.083 0.085 0.080 0.049 0.052 0.054 0.047 0.052 0.052 0.054 0.057 0.054 0.009 0.007 0.014 0.016R 0.085 0.088 0.083 0.049 0.052 0.052 0.047 0.052 0.052 0.054 0.057 0.054 0.014 0.012 0.019 0.021 0.005S 0.087 0.090 0.085 0.054 0.057 0.059 0.052 0.057 0.057 0.059 0.061 0.059 0.005 0.007 0.019 0.012 0.005 0.009T 0.083 0.085 0.080 0.052 0.054 0.056 0.049 0.054 0.054 0.056 0.059 0.056 0.009 0.007 0.016 0.019 0.005 0.005 0.005U 0.087 0.089 0.084 0.054 0.056 0.059 0.052 0.056 0.056 0.059 0.061 0.054 0.058 0.014 0.012 0.019 0.021 0.002 0.005 0.007

Fig. 3. Single most parsimonious circle tree for 21 Heterotermes haplotypes by using a branch and bound of PAUP*(Swofford 2001). Bootstrap values for 1000 replicates are listed above the branches supported at �50%.

562 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 97, no. 3

Hedges 1996a,b, 2001 andHedgeset al. 1992, 1994haveargued that the fauna of the region shows a pattern ofconcordant dispersal from South America. Numerousothers have argued for taxonomic, and more recently,phylogenetic congruence pointing toward a vicariantorigin of theCaribbean fauna (Rosen 1976, 1985; Lieb-herr 1988; Page and Lydeard 1994; Crother andGuyer1996; Guyer and Crother 1996). Haplotype groups Iand V could be persuasively argued either way. Hap-lotype group IV (The Bahamas) was also distinct andwas, because of proximity, presumed the likely sourceof introduction of Heterotermes into Miami, FL. How-ever, results of our analysis can conclusively rule outthis assumption.Heterotermes from Veracruz, Mexico,and Xunatunich ruins, Belize, were clearly distinctfrom all Caribbean groups. The only endemic speciesto the continental United States,H. aureus from SantaRita, AZ,was also unique. Although the exact origin ofH. aureus is unknown, the island arc model for thePaciÞc origin of American terrains at the western andeastern boundary of a former Philippine-like easternPaciÞcplate (“Cordilleria”) that progressivelymergedwithNorth, South, andCentral America betweenMe-sozoic and Tertiary time (Moores 1998) may be oneexplanation (Grehan 2001). Because the only en-demic species of this genus known to occur in theUnited States isH. aureus, in the southwestern UnitedStates and Mexico, we elected to include this sampleinto the study to clarify its relationship with its Ca-ribbean cousins (Fig. 2). Clearly, H. aureus is distinctfromCaribbean relatives andwasmost closely related

to our sample from Veracruz, Mexico, and the Xuna-tunich ruins, Belize. Haplotype group III (Turks andCaicos islands and St. Barthelemy) was a disjunctgroup represented in this study and supports eitherover-water dispersal or an introduction of anthropo-genic origin. We suggest that the source of an anthro-pogenic introduction of Heterotermes into Miami, FL,is themost likely from thehaplotypegroup II (Jamaicaand Grand Cayman) characterized in our study. Adetailed population genetics analysis of group II Het-erotermes is warranted to further support this hypoth-esis.Mechanisms for over-water dispersal of Heterot-

ermes, however, must also include shipboard infesta-tions during some two to over four centuries of inter-island trade and, more recently, pleasure boat trafÞc,thus obscuring the natural forces of island speciesdiversity as reported by MacArthur and Wilson(1967). Human-aided establishment of Heterotermeshas been conÞrmed for Florida (Scheffrahn and Su1995) as well as Madagascar, Mauritius, and the mid-Atlantic island of St. Helena where its origin wasblamed on an infested ship dismantled there in�1840(Gay 1967). Data herein suggest that on small, ratherisolated islands with active seaports such as GrandTurk, St. Barthelemy, and Grand Cayman, theHetero-termes populations are not endemic. Larger islandswith little present or historic ship trafÞc such as Bar-buda, Mayaguana, and the central Caicos islands haveno Heterotermes populations. Based on habitat typesalone,Heterotermespopulations shouldbe able to exist

Fig. 4. Topology obtained by maximum-likelihood analysis based on the HYK85 model (see text) for 21 West IndianHeterotermes haplotypes. Log L � �1461.35245.

May 2004 SZALANSKI ET AL.: Heterotermes GENETICS 563

on all islands in the West Indies. Competition fromother endemic rhinotermitid species is limited or non-existent in the West Indies.Based on genetic haplotypes, there were represen-

tatives within the current study that did not conformto the more visible population structures observed.For example, Heterotermes collected from Barbados,Martinique, and Monos (Trinidad) Island exhibitedgreater genetic similarity to Heterotermes from groupV. Likewise, Heterotermes from Bonaire were moresimilar to those found in group II (Figs. 3 and 5).Group VI, representing Antigua, St. Maarten, PuertoRico, and St. Kitts, was overlapped by group V. Themost plausible explanation for the discordant geneticstructure observed in these instances would implyshipborne introductions, but over-water dispersal bynatural means cannot be ruled out.The mitochondrial haplotype patterns observed in

these instanceswould conform to category I of AviseÕsphylogeographic model (Avise et al. 1987). CategoryI, characterized by phylogenetic discontinuities withspatial separation, is one of the most commonly en-countered in studies that encompass large geographiczones as presented in this study.The anthropogenic source of Heterotermes in Flor-

ida could not be determined on morphologicalgrounds alone (Scheffrahn and Su 1995). Our geneticanalysis ofHeterotermes from theWest Indies suggeststhat the likely origin of this nonendemic pest to Flor-ida was either Jamaica or Grand Cayman. Endemic

populations of Heterotermes do not exist in Floridaprobably due to its submergence during interglacialperiods of the Pleistocene (Webb 1990). However,anthropogenic introductions of exotic termite speciesto south Florida and elsewhere are likely to continue.For this reason, identifying where these infestationsoriginate is crucial to limiting their establishment andimplementing possible interdiction policies that couldcurtail their introductions. It has already been dem-onstrated that other subterranean termites can extendthemselves well beyond what would be consideredtheir normal habitation range. Reticulitermes termiteshave been introduced into Canada, France, England,Germany, and South America where they have estab-lished themselves as serious structural pests (Austin etal. 2002). Other recent discoveries of pestiferous ter-mite species introduced into Florida include Coptot-ermes havilandi (�C. gestroi; Su et al. 1997, Kirton andBrown 2003) and Nasutitermes costalis (Holmgren)(Scheffrahn et al. 2002). IdentiÞcation through mo-lecular diagnostics (Szalanski et al. 2003), and tracingthe origins of exotically introduced species throughthe use of various genetic analyses will become anincreasingly important component to interdicting un-wanted pests like Heterotermes. In light of the resultsof this study, attention should be given to the focalpoints from where these species originate to see whatregulatory measures should be enacted to stem theßowof the destructive termites into theUnited States.

Fig. 5. Geographical distribution of West Indian Heterotermes haplotype groupings and species.

564 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 97, no. 3

Acknowledgments

We James A. Chase and John R. Mangold (Terminix In-ternational) and Boudanath Maharajh and Paul Ban (Uni-versity of Florida) for assistance in collecting the Heterot-ermes samples used in this study. Research was supported inpart by the University of Arkansas, Arkansas AgriculturalExperiment Station.

References Cited

Adamson, A. M. 1938. Notes on termites destructive tobuildings in the Lesser Antilles. Trop. Agric. 15: 220Ð224.

Austin, J. W., A. L. Szalanski, P. Uva, A. G. Bagneres, and A.Kence. 2002. A comparative genetic analysis of the sub-terranean termite genus Reticulitermes (Isoptera: Rhino-termitidae). Ann. Entomol. Soc. Am. 95: 753Ð760.

Austin, J. W., A. L. Szalanski, R. E. Gold, and B. T. Foster.2004. Genetic variation and geographical distribution ofthe subterranean termite genus Reticulitermes in Texas.Southwest. Entomol. (in press).

Austin, J. W., A. L. Szalanski, and B. J. Cabrera. 2004. Aphylogenetic analysis of the subterranean termite familyRhinotermitidae (Isoptera) using the mitochondrial cy-tochrome oxidase (COII) gene. Ann. Entomol. Soc. Am.(in press).

Avise, J. C., J. Arnold, R. M. Ball, E. Bermingham, T. Lamb,J. E. Neigel, C. A. Reeb, and N. C. Saunders. 1987. In-traspeciÞc phylogeography: the mitochondrial DNAbridge between population genetics and systematics.Annu. Rev. Ecol. Syst. 18: 489Ð522.

Clement, J. L., A. G. Bagneres, P. Uva, L. Wilfert, A. Quin-tana, J. Reinhard, and S. Dronnet. 2001. Biosystematicsof Reticulitermes termites in Europe: morphological,chemical and molecular data. Insectes Soc. 48: 202Ð215.

Constantino, R. 1998. Catalog of the living termites of theNew World (Insecta: Isoptera). Arq. Zool. (Sao Paulo)35: 135Ð231.

Constantino, R. 2000. Key to the soldiers of South Amer-ican Heterotermes with a new species from Brazil(Isoptera: Rhinotermitidae). Insect Syst. Evol. 31: 463Ð472.

Crother, B. I., and C. Guyer. 1996. Caribbean historical bio-geography: was the dispersal-vicariance debate elimi-nated by an extraterrestrial bolide? Herpetologica 52:440Ð465.

Donovan, S. E., D. T. Jones, W. A. Sands, and P. Eggleton.2000. Morphological phylogenetics of termites(Isoptera). Biol. J. Linn. Soc. 70: 467Ð513.

Emerson, A. E. 1925. The termites of Kartabo Barica Dis-trict, British Guiana. Zoologica 6: 291Ð459.

Emerson, A. E. 1971. Tertiary fossil species of the Rhinot-ermitidae (Isoptera), phylogeny of genera, and recipro-cal phylogenyof associatedFlagellata (Protozoa) and theStaphylinidae (Coleoptera). Bull. Am. Mus. Nat. Hist.146: 243Ð304.

Felsenstein, J. 1985. ConÞdence limits on phylogenies: anapproach using the bootstrap. Evolution 39: 783Ð791.

Gay, F. J. 1967. A world review of introduced species oftermites. CSIRO, Melbourne, Bull 286.

Grehan, J. 2001. Biogeography and evolution of theGalapa-gos: integration of the biological and geological evidence.Biol. J. Linn. Soc. 74: 267Ð287.

Guyer, C., and B. I. Crother. 1996. Additional comments onthe origin of the West Indian herpetofauna. Herpeto-logica 52: 620Ð622.

Hagen, H. A. 1858. Specielle Monographie der Termiten.Linn. Entomol. 12: 4Ð342.

Hall, T. A. 1999. BioEdit: a user-friendly biological se-quence alignment [ed.], and analysis program for Win-dows 95/98/NT. Nucleic Acids. Symp. Ser. 41: 95Ð98.

Hedges, S. B. 1996a. Historical biogeography ofWest Indianvertebrates. Annu. Rev. Ecol. Syst. 27: 163Ð196.

Hedges, S. B. 1996b. More on West Indian zoogeography.Herpetologica 52: 622Ð624.

Hedges, S. B. 2001. Biogeography of the West Indies: anoverview, pp. 15Ð33. In C. A. Woods, and F. E. Sergile[eds.], Biogeography of the West Indies, patterns andperspectives. CRC, Boca Raton, FL.

Hedges, S. B., C. A.Hass, andL. R.Maxson. 1992. Caribbeanbiogeography: molecular evidence for dispersal in WestIndian terrestrial vertebrates. Proc. Nat. Acad. Sci. U.S.A.89: 1909Ð1913.

Hedges, S. B., C. A. Hass, and L. R. Maxson. 1994. Reply:towards a biogeography of the Caribbean. Cladistics 10:43Ð55.

Husseneder, C., and J. K. Grace. 2001. Similarity is relative:hierarchy of genetic similarities in the Formosan subter-ranean termite (Isoptera: Rhinotermitidae) in Hawaii.Environ. Entomol. 30: 262Ð266.

Jenkins, T. M., C. J. Basten, R. Dean, S. E. Mitchell, S.Kresovich, and B. T. Forschler. 1998. Matriarchal ge-netic structure of Reticulitermes (Isoptera: Rhinotermiti-dae) populations. Sociobiology 33: 239Ð263.

Jenkins, T. M., C. J. Basten, S. Kresovich, and B. T. Forschler.1999. Mitochondrial gene sequence questions Reticuli-termes sp. social structure (Isoptera: Rhinotermitidae).Sociobiology 34: 161Ð172.

Jenkins, T. M., R. E. Dean, and B. T. Forschler. 2002. DNAtechnology, Interstate commerce, and the likely origin ofFormosan subterranean termite (Isoptera: Rhinotermiti-dae) infestation inAtlanta, Georgia. J. Econ. Entomol. 95:381Ð389.

Kambhampati, S., and P. T. Smith. 1995. PCR primers forthe ampliÞcation of four insect mitochondrial gene frag-ments. Insect Mol. Biol. 4: 233Ð236.

Kambhampati, S., K. M. Kjer, and B. L. Thorne. 1996. Phy-logenetic relationship among termite families based onDNA sequence of mitochondrial 16S ribosomal RNAgene. Insect Mol. Biol. 5: 229Ð238.

Kimura, M. 1980. A simple method for estimating evolu-tionary rate of base substitutions through comparativestudy of nucleotide sequences. J. Mol. Evol. 16: 111Ð120.

Kirton, L. G., and V. K. Brown. 2003. The taxonomic statusof pest species ofCoptotermes in Southeast Asia: resolvingthe paradox in the pest status of the termitesCoptotermesgestroi, C. havilandi, and C. travians (Isoptera: Rhinoter-mitidae). Sociobiology 42: 43Ð63.

Liebherr, J. K. 1988. Biogeographic pattens of West IndianPlatynus carabid beetles (Coleoptera), pp. 77Ð140. In J. K.Liebherr [ed.], Zoogeography of Caribbean insects.Comstock, Ithaca, NY.

Light, S. F. 1933. Termites of western Mexico. Univ. Calif.Public Entomol. 6: 79Ð152, �11 plates.

Marini, M., and B. Mantovani. 2002. Molecular relation-ships among European samples of Reticulitermes(Isoptera, Rhinotermitidae). Mol. Phylogenet. Evol. 22:454Ð459.

MacArthur, R. H., and E. O. Wilson. 1967. The theory ofisland biogeography, pp. 123Ð144. Princeton UniversityPress, Princeton, NJ.

Moores, E. M. 1998. Ophiolites, the Sierra Nevada, “Cordil-leria”, and orogeny along the PaciÞc and Caribbean mar-gins of North and South America. Int. Geol. Rev. 40:40Ð54.

May 2004 SZALANSKI ET AL.: Heterotermes GENETICS 565

Page, R.D.M., and C. Lydeard. 1994. Towards a cladisticbiogeography of the Caribbean. Cladist 10: 41Ð41.

Rosen, D. E. 1976. A vicariance model of Caribbean bioge-ography. Syst. Zool. 24: 431Ð464.

Rosen, D. E. 1985. Geological hierarchies and biogeo-graphic congruence in the Caribbean. Ann.Missouri Bot.Garden 72: 636Ð659.

Scheffrahn, R. H., and N.-Y. Su. 1995. A new subterraneantermite introduced to Florida: Heterotermes Froggatt(Rhinotermitidae: Heterotermitinae) established in Mi-ami. Fla. Entomol. 78: 623Ð627.

Scheffrahn, R. H., J.P.E.C. Darlington, M. S. Collins, J. Kre-cek, and N.-Y. Su. 1994. Termites (Isoptera: Kalotermiti-dae, Rhinotermitidae, Termitidae) of the West Indies.Sociobiology 24: 213Ð238.

Scheffrahn, R. H., J. A. Chase, J. R. Mangold, J. Krecek, andN.-Y. Su. 1999. First record of Reticulitermes (Isoptera:Rhinotermitidae) from the West Indies: R. flavipes onGrand Bahama Island. Fla. Entomol. 82: 480Ð482.

Scheffrahn, R.H., B. J. Cabrera,W.H.Kern, Jr., andN.-Y. Su.2002. Nasutitermes costalis (Isoptera: Termitidae) inFlorida: Þrst record of a non-endemic establishment by ahigher termite. Fla. Entomol. 85: 273Ð275.

Scheffrahn R. H., S. C. Jones, J. Krecek, J. A. Chase, J. R.Mangold, and N-Y. Su. 2003a. Taxonomy, distribution,and notes on the termites (Isoptera: Kalotermitidae, Rhi-notermitidae, Termitidae) of Puerto Rico and the U.S.Virgin Islands. Ann. Entomol. Soc. Am. 96: 181Ð201.

Scheffrahn R. H., J. Krecek, B. Maharajh, J. A. Chase, J. R.Mangold and C. K. Starr. 2003b. Termite Fauna(Isoptera) of Trinidad & Tobago,West Indies, pp. 33Ð38.Occ. Papers. Dept. Life Sci. Univ. West Indies, St. Au-gustine, Trinidad & Tobago No. 12.

Simon, C., F. Frati, A. Beckenbach, B. Crespi, H. Liu, and P.Flook. 1994. Evolution, weighting, and phylogeneticutility of mitochondrial gene sequences and a compila-tion of conserved polymerase chain reaction primers.Ann. Entomol. Soc. Am. 87: 651Ð701.

Snyder,T.E. 1924. Descriptionsofnewspecies andhithertounknown castes of termites from America and Hawaii.Proc. U.S. Natl. Mus. no 2496. 64: 1Ð39 � plates.

Snyder, T. E. 1949. Catalog of the termites (Isoptera) of theworld. Smith. Misc. Collect. 112: 1Ð490.

Snyder, T. E. 1956. Termites of the West Indies, the Baha-mas, and Bermuda. J. Agric. Univ. Puerto Rico. 40: 189Ð202.

Su, N.-Y., R. H. Scheffrahn, and T. Weissling. 1997. A newintroduction of a subterranean termite,Coptotermes havi-landi Holmgren (Isoptera: Rhinotermitidae) in Miami,Florida. Fla. Entomol. 80: 408Ð411.

Swofford, D. L. 2001. PAUP*: phylogenetic analysis usingparsimony (*and other methods), version 4.0b10. Si-nauer, Sunderland, MA.

Szalanski, A. L., D. S. Sikes, R. Bischof, and M. Fritz. 2000.Population genetics andphylogenetics of the endangeredAmerican burying beetle, Nicrophorus americanus (Co-leoptera: Silphidae).Ann.Entomol. Soc.Am. 93: 589Ð594.

Szalanski, A. L., J. W. Austin, and C. B. Owens. 2003. Iden-tiÞcation of Reticulitermes spp. (Isoptera: Reticuliterma-tidae) fromSouthCentral United States by PCR-RFLP. J.Econ. Entomol. 96: 1514Ð1519.

Tajima, F., and M. Nei. 1984. Estimation of evolutionarydistance between nucleotide sequences. Mol. Biol. Evol.1: 269Ð285.

Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994.CLUSTAL W: improving the sensitivity of progressivemultiples sequencealignments throughsequenceweight-ing, position-speciÞc gap penalties and weight matrixchoice. Nucleic Acids Res. 22: 4673Ð4680.

Thompson, G. J., O. Kitade, N. Lo, and R. H. Crozier. 2000.Phylogenetic evidence for a single, ancestral origin of aÔtrueÕ worker caste in termites. J. Evol. Biol. 13: 869Ð881.

Webb, S. D. 1990. Historical biogeography, pp. 70Ð100. InR. L. Myers, and J. J. Ewel [eds.], Ecosystems of Florida.University of Central Florida Press, Orlando, FL.

Woods,C. A., andF.E. Sergile [eds]. 2001. Biogeography oftheWest Indies: patterns and perspectives, 2nd ed., CRC,Boca Raton, FL.

Received 8 September 2003; accepted 9 December 2003.

566 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 97, no. 3