Bulletin of Entomological Research (2002) 92, 337–341 DOI: 10.1079/BER2002168 PCR–RFLP identification of endemic Chilean species of Rhagoletis (Diptera: Tephritidae) attacking Solanaceae M. Salazar 1 , C. Theoduloz 1 , A. Vega 1 , F. Poblete 1 , E. González 1 , R. Badilla 2 and L. Meza-Basso 1 * 1 Instituto de Biología Vegetal y Biotecnología, Universidad de Talca, Casilla 747, Talca, Chile: 2 Servicio Agrícola y Ganadero, VII Región, Talca, Chile Abstract Four species of the genus Rhagoletis are native to Chile: R. nova (Schiner), R. conversa, (Brèthes), R. penela Foote and R. tomatis Foote. Currently, identification of these species is based on morphological criteria, but their strong similarity makes precise recognition difficult. To clarify species separation for quarantine purposes, a reliable method based on a PCR–RFLP procedure is reported. A DNA region containing mitochondrial NADH dehydrogenase genes was selected as a target sequence for the analysis. The amplification products (c. 1 kb) were digested with either SspI or DdeI, yielding specific patterns that differentiated each of the endemic species. Complete nucleotide sequences were determined, confirming empirical restriction maps. This report updates information on the geographical distribution of Rhagoletis species in Chile. Introduction The Tephritidae, commonly known as fruit flies, is one of the largest families of Diptera with over 500 genera and 4000 species described so far (Thompson, 1999). Most species do not infest fruits and are economically harmless. However, some species belonging to the genera Anastrepha, Bactrocera, Ceratitis, Dacus and Rhagoletis (Boller & Prokopy, 1976) cause severe constraints on agricultural production, becoming pests both within and beyond their native boundaries. As a result, quarantine restrictions have been imposed by many countries to limit further spread of fruit fly pests around the world. Tomato producers in Chile are interested in developing foreign markets, but the four Chilean Rhagoletis species (R. nova (Schiner), R. conversa (Brèthes), R. penela Foote and R. tomatis Foote) infest fruit of the Solanaceae and create quarantine difficulties. These species belong to the nova group and are endemic to Chile, except for R. tomatis which is also found in neighbouring Peru (Frías & Martínez, 1991). From a phytosanitary and economic point of view, R. nova and R. tomatis are most important due to their damage to fruits of sweet cucumber Solanum muricatum (Aiton) (Solanaceae) and tomato Lycopersicon esculentum (Mill) (Solanaceae), respectively (Frías, 1992). Current evidence indicates that R. conversa and R. penela are potentially less important as their attacks are confined to wild Solanaceae (Frías, 1992). The identification of these species relies on morphological criteria (Frías & Martínez, 1991). However, R. nova and R. conversa are morphologically very similar (Foote, 1981; Frías & Martínez, 1991). Accurate identification of Rhagoletis specimens is essential for efficient quarantine practices and the development and deployment of pest management strategies. In order to reduce the chances of a false identification of morphologically similar species, methods that enable genotype-based identification may be employed (Loxdale & Lushai, 1998; Benecke & Wells, 2000). DNA amplification via the polymerase chain reaction (PCR) has become very popular in modern systematics because of its simplicity and specificity. In insect systematics, the most commonly used mitochondrial regions correspond to the cytochrome oxidase genes, 16S ribosomal DNA and cytochrome b (Caterino et al., 2000). Less attention has been *Author for correspondence Fax: 56 71 200279 E-mail: mezabas@pehuenche.utalca.cl.