HORTICULTURAL ENTOMOLOGY Polymerase Chain Reaction-Restriction Fragment-Length Polymorphism Method to Distinguish Liriomyza huidobrensis from L. langei (Diptera: Agromyzidae) Applied to Three Recent Leafminer Invasions SONJA J. SCHEFFER, 1 ANURA WIJESEKARA, 2 DIEDRICH VISSER, 3 AND REBECCA H. HALLETT 4 J. Econ. Entomol. 94(5): 1177Ð1182 (2001) ABSTRACT A molecular method is presented for differentiating the morphologically cryptic leafminers Liriomyza langei Frickand L. huidobrensis (Blanchard).Thismethodrequirespolymerase chain reaction (PCR) ampliÞcation of a 1031-bp region of mitochondrial cytochrome oxidase DNA followed by restriction fragment analysis using the restriction enzymes SpeI and EcoRV. SpeI cuts the mitochondrial fragment of L. langei into two fragments, but does not cut the L. huidobrensis fragment. EcoRV cuts the L. huidobrensis fragment into two fragments, but does not cut the L. langei fragment. This PCR-restriction fragment-length polymorphism (RFLP) method is faster and less costlythanDNAsequencing,whichiscurrentlytheonlyotherwaytodifferentiatethesetwospecies. We apply the method to samples from recently introduced leafminer populations in Sri Lanka, Canada, and South Africa and Þnd that the invasive leafminer in all three locations is L. huidobrensis. KEY WORDS Agromyzidae, polymerase chain reaction-restriction fragment-length polymor- phism, molecular identiÞcation, pea leafminer, introduced species, invasive species THE LEAFMINING FLIES Liriomyza huidobrensis (Blan- chard) and L. langei Frick are important pests of a wide variety of vegetable and ßower crops (Spencer 1973, Steck 1996). In 1973, Spencer (1973) synony- mized L. langei with L. huidobrensis because of an apparent lack of morphological differences separating the two species. However, recent phylogenetic anal- yses of both mitochondrial and nuclear DNA se- quence data have shown that these two groups of leafminers are substantially divergent in molecular characters and therefore represent morphologically cryptic species (Scheffer 2000, Scheffer and Lewis 2001). Scheffer and Lewis (2001) resuscitated the name L. langei for the cryptic species found in Cali- fornia and Hawaii and restricted the name L. huido- brensis to the cryptic species found in South and Cen- tral America. Although L. huidobrensis and L. langei are endemic to the New World, since 1989 one or both of these species has spread to a number of additional regions including Europe (Cheek et al. 1993, Weintraub and Horowitz 1995), the Middle East (Weintraub and Horowitz 1995), and Asia (Shepard et al. 1996). Avail- able evidence suggests that in most cases the invading ßies are L. huidobrensis spreading from South and/or Central America. In some cases, importation of in- fested plant material from South or Central America has been implicated in new infestations (de Goffau 1991, Bartlett 1993). Additionally, analysis of both mi- tochondrial and nuclear DNA sequence data has un- ambiguously shown that samples from invasive pop- ulations in Israel, Indonesia, and Sri Lanka belong to L. huidobrensis rather than L. langei (Scheffer 2000, Scheffer and Lewis 2001). To date, L. langei has not been detected in any geographic location other than North America and Hawaii. However, given that it is only recently that this species could be distinguished from L. huidobrensis, it is possible that invasive pop- ulations of L. langei arepresentinotherareas.Because L. huidobrensis and L. langei are suspected to differ in preferred hosts and in insecticide resistance status (Bartlett 1993, Weintraub and Horowitz 1995), it is important for management efforts that the species identity of newly introduced populations be known. Currently, no morphological differences are known to differentiate L. langei from L. huidobrensis, al- though morphological studies are currently under- way. Species identity can be readily determined using DNAsequencedatafromanyofseveralmitochondrial and nuclear genes (Scheffer 2000, Scheffer and Lewis 2001), but this method is somewhat time-consuming and expensive for those not routinely involved with DNA sequencing. The purpose of this article is to present a less ex- pensive molecular method that can be used to rapidly differentiate L. langei from L. huidobrensis. This method uses the PCR combined with RFLP analysis 1 Systematic Entomology Laboratory, Agriculture Research Ser- vice, U.S. Department of Agriculture, Building 005, Room 137, BARC-W, 10300 Baltimore Avenue, Beltsville, MD 20705 (e-mail: sscheffe@sel.barc.usda.gov). 2 Division of Entomology, Horticultural Crops Research and De- velopment Institute, P.O. Box 11, Peradeniya, 20400, Sri Lanka. 3 ARC-Roodeplaat Vegetable and Ornamental Plant Institute, Pri- vate Bag X293, Pretoria, 001, South Africa. 4 Department of Environmental Biology, Ontario Agricultural Col- lege, University of Guelph, Guelph, ON, Canada N1G 2W.