THE UTILITY OF THE INTERNAL TRANSCRIBED SPACER REGION 2 (ITS2) IN CONFIRMING SPECIES BOUNDARIES IN THE GENUS GONATOCERUS: COMPARISON TO THE CYTOCHROME OXIDASE SUBUNIT I (COI) GENE AND TAXONOMIC DATA: MOLECULAR KEY BASED ON ITS2 SIZES Project Leaders:

We sequenced the nuclear ribosomal internal transcribed spacer region 2 (ITS2) from several glassy-winged sharpshooter (GWSS) [Homalodisca vitripennis Germar (=H. coagulata Say)] egg parasitoid species (Hymenoptera: Mymaridae) belonging to the genus Gonatocerus Nees to test the utility of this fragment to confirm species boundaries and to define phylogenetic relationships. A total of 35 specimens belonging to 10 named species, one unnamed species, and two specimens from another mymarid genus (Anagrus erythroneurae) (outgroup) were analyzed. A phylogenetic tree generated using the neighbor-joining algorithmic method showed that each named Gonatocerus species formed its own unique taxonomic unit or clade with very strong bootstrap support (100%), confirming species boundaries. The ITS2 fragment confirmed species boundaries as well as cytochrome oxidase subunit I (COI) sequence data. Furthermore, the phylogenetic relationships among species generated by the ITS2 fragment were in excellent agreement with those delineated by taxonomic data. The current results clearly confirm the utility of the ITS2 fragment in confirming species boundaries of egg parasitoids beloning to the genus Gonatocerus. The results showed that the ITS2 fragment appears to be phylogenetically more informative or valuable than that inferred by COI sequence data. Since several important Gonatocerus species were analyzed, a molecular key based on ITS2 sizes was developed. In the event two species (e. g., G. ashmeadi and G. metanotalis and G. walkerjonesi and G. annulicornis) were found with similarly sized ITS2 fragments, inter-simple sequence repeat-polymerase chain reaction (ISSR-PCR) DNA fingerprinting was performed to distinguish them. ISSR-PCR very clearly distinguished the aforementioned species, demonstrating that it is an excellent molecular diagnostic tool. The current results are important to the biological control program in California. INTRODUCTION Accurately identifying natural enemies is critical to the success of classical biological control programs and lack of proper identification procedures has affected several projects (Messing and Aliniazee 1988, Löhr et al. 1990, Narang et al. 1993). Among others, DNA markers such as the nuclear ribosomal internal transcribed spacer regions (e. g., ITS2) are used to characterize parasitoid taxa because these DNA regions usually evolve relatively rapidly (Hillis and Dixon 1991, Narang et al. 1993). The ITS regions have been used extensively in the examination of the taxonomic status of species and for diagnostic purposes (Collins and Paskewitz 1996, Stouthamer et al. 1999). Many examples of phylogenetic studies inferred by the nuclear ITS regions or fragments, including different sized fragments, exists in the literature (Marinucci et al. 1999, Förster et al. 2000, Pryor and Gilbertson 2000, Alvarez and Hoy 2002, Thomson et al. 2003, de León et al. 2006a, Wagener et al. 2006), including those by Stouthamer et al. (1999). OBJECTIVES Sequence the nuclear ribosomal internal transcribed spacer region 2 (ITS2) from several GWSS egg parasitoid species (11) belonging to the genus Gonatocerus to test the utility of this rDNA fragment to: 1) confirm species boundaries and 2) define phylogenetic relationships. RESULTS AND CONCLUSIONS Species boundaries inferred by the ribosomal internal transcribed spacer region 2 (ITS2) We obtained 8 of the 13 named Gonatocerus Nees species delineated by Triapitsyn (2006) and Triapitsyn et al. (2006) and several named and one unnamed species from South America for a total of 11 species. A total of 35 ingroup specimens were analyzed and two specimens from Anagrus erythroneurae Trjapitzin & Chiappini (also a mymarid species) were included as an outgroup. Each named Gonatocerus species formed its own taxonomic unit or distinct clade (Figure 1), corroborating the species boundaries of Triapitsyn (2006) and Triapitsyn et al. (2006). A neighbor-joining distance tree showed that each taxonomic unit was supported by very strong bootstrap values, in fact, each received 100% support. In addition, the unnamed Gonatocerus species (G. sp. 6) from Argentina also clustered into its distinct clade, suggesting that it is a separate or valid species. Analysis of several other Gonatocerus species inferred by the ITS2 DNA fragment are in progress to complete this project. As previously demonstrated by Vickerman et al. (2004) and Triapitsyn et al. (2006) no divergence or differences were seen in the five geographic populations [California, Texas (Weslaco and San Antonio), Florida, and Louisiana] of G. ashmeadi, as they all formed their unique clade.