OF GONATOCERUS TUBERCULIFEMUR FROM SOUTH AMERICA UNCOVERS DIVERGENT CLADES : PROSPECTIVE EGG PARASITOID CANDIDATE AGENT FOR THE GLASSY-WINGED SHARPSHOOTER IN CALIFORNIA Project Leaders :

In present study we genetically characterized the prospective South American egg parasitoid candidate, Gonatocerus tuberculifemur (Ogloblin) of the glassy-winged sharsphooter (GWSS), Homalodisca vitripennis (Germar) [=H. coagulata (Say)] for a neoclassical biological control program in California. Two molecular methods, inter-simple sequence repeatpolymerase chain reaction (ISSR-PCR) DNA fingerprinting and a phylogeographic approach inferred by the mitochondrial cytochrome oxidase subunit I gene (COI). Five geographic populations from South America were analyzed; in addition, a phylogenetic analysis was performed with several named and two unnamed Gonatocerus Nees species. DNA fingerprinting uncovered a fixed geographic banding pattern difference in the population from San Rafael, Mendoza Province, Argentina. The COI analysis uncovered haplotype or geographic structure in G. tuberculifemur. A neighbor-joining distance tree clustered the populations into two well-supported distinct clades with very strong bootstrap values (96-100%) with the population from San Rafael clustering into a separate clade than the rest of the South American populations. No haplotype sharing was observed between individuals from the two clades. A phylogenetic analysis performed by the neighbor-joining method of 15 Gonatocerus Nees species confirmed species boundaries and again uncovered two distinct clades in G. tuberculifemur with very strong bootstrap support (96-100%). The two molecular methods were in accord and the evidence is suggestive of a species level divergence. Because G. tuberculifemur is under consideration as a potential biological control agent for the invasive GWSS in California, understanding possible cryptic variation of this species is critical. INTRODUCTION Uncertainty exists as to whether egg parasitoids native to California will be as effective against the glassy-winged sharpshooter (GWSS) Homalodisca vitripennis (Germar) [=H. coagulata (Say)]as they are in their co-evolved native range (Jones 2001, Logarzo et al. 2003, 2004, Virla et al. 2005). As a consequence, beginning in 2000, egg parasitoids of closely related hosts belonging to the sharsphooter tribe Proconiini [Tapajosa rubromarginata (Signoret)] were sought from regions in South America where climate types and habitats were similar to California for a neoclassical biological control program (Jones 2001, Logarzo et al. 2005). In surveys conducted in Argentina and Chile during 2000 through 2005, two prospective egg parasitoid candidate agents were identified among several Gonatocerus Nees species reared from T. rubomarginata (Jones et al. 2005, Logarzo et al. 2005, Virla et al. 2005). The egg parasitoid candidates from South America were identified by S. V. Triapitsyn (UC-Riverside) as Gonatocerus tuberculifemur and G. metanotalis (Ogloblin) (Hymenotpera: Mymaridae). Gonatocerus tuberculifemur is now being permitted for release in California (CDFA 2005). Mymarid wasps are the best-known egg parasitoids for controlling populations of leafhoppers (Huber 1986, Döbel and Denno 1993). Molecular studies of insects are becoming increasingly important in resolving taxonomic relationships critical to the success of biological control programs. Identifying the correct natural enemy is critical to the success of classical biological control programs. Lack of proper identification procedures has affected several projects (Messing and Aliniazee 1988, Löhr et al. 1990, Narang et al. 1993). OBJECTIVE The aim of the present study was to survey molecular methods useful in egg parasitoid identification and discrimination and investigate the possibility that G. tuberculifemur (Ogloblin) could exist as a cryptic species complex. In addition, perform a phylogenetic analysis with several species within the genus Gonatocerus Nees to confirm species boundaries and to test the support for the species groups considered. RESULTS AND CONCLUSIONS ISSR-PCR DNA fingerprinting. Amplification reactions were performed with geographic populations from Argentina and Chile with 5-9 separate individuals from pooled egg masses per location. Locations included were: Argentina: Rio Colorado (RC) (Rio Negro Province), San Rafael (SR) (Mendoza), San Miguel de Tucumán (SMT) (Tucumán), and Chile: Jalsuri (CH). Previously, we have demonstrated a positive correlation between ISSR-PCR banding patterns and species distinction (de León and Jones 2004, de León et al. 2004a,b, 2006). In addition, we have utilized the method to distinguish about 8 Gonatocerus species. As a first approach, we asked whether the ISSR-PCR method was suitable to distinguish geographic populations of G. tuberculifemur from Argentina and Chile. The results of this analysis are shown on Figure 1. Three geographicor population-specific bands were identified within the San Rafael population, as indicated by the arrows. Slight variation was seen within the rest of the populations, but in general, similar banding patterns were observed within these