Proceedings of 6th International Fruit Fly Symposium 6–10 May 2002, Stellenbosch, South Africa pp. 439–443 A phylogenetic study of the family Tephritidae (Insecta: Diptera) using a mitochondrial DNA sequence P. Fernández, D. Segura, C. Callejas & M.D. Ochando* Departamento de Genética, Facultad de Ciencias Biológicas, Universidad Complutense, 28040 – Madrid, Spain Achievements in tephritid taxonomy have greatly contributed to both basic research and pest management programmes. However, despite the large amount of taxonomic data available, the higher classification of the family Tephritidae is still a matter of debate. A molecular approach could help to provide a more accurate classification. A molecular study was therefore undertaken to gain insight into the phylogenetic relationships within the family Tephritidae. A DNA region of the mitochondrial cytochrome oxidase II gene was compared in species representing six genera of the family, namely Ceratitis, Rhagoletis, Dacus, Bactrocera, Anastrepha and Toxotrypana. A dendrogram was constructed using the neighbour-joining method with Liriomyza huidobrensis and Drosophila yakuba as outgroups. Two main clusters were obtained in the tree, the first grouping being the Ceratitis species, C. capitata, C. rosa, and C. cosyra, and the second showing two main branches, one for Dacus, Bactrocera and Rhagoletis, and the other for Anastrepha and Toxotrypana. The results are discussed in relation to published phylogenies. INTRODUCTION a better understanding of the phylogenetic rela- Among the most devastating of agricultural tionships within the Tephritidae family (Han & pests, the family Tephritidae, commonly known as McPheron 1994, 1997, 2001; Malacrida et al. 1996; fruit flies, includes more than 4000 species in McPheron & Han 1997; Smith & Bush 1997; some 500 genera distributed all around the world Morrow et al. 2000; Han 2000). (White & Elson-Harris 1994). The enormous eco- Specifically, mitochondrial DNA is a powerful nomic effort required in eradication programmes, material for phylogenetic studies. Its small size, together with great crop losses that these flies different rates of evolutionary change, lack of cause in fruit production, explains the increasing recombination, and maternal inheritance make it interest in the study of their biology. suitable material for systematic studies at a wide Taxonomy is an essential foundation of biologi- variety of taxonomic levels (Simon et al. 1994; Han cal research. Developments in tephritid taxon- & McPheron 2001). Among the mitochondrial omy have greatly contributed to progress in genes, cytochrome oxidase II, usedbydifferent areas of pure research and to pest management authors to infer relationships within insect species programmes. However, despite the large number (Sperling & Hickey 1994; Caterino & Sperling of taxonomic data available, the higher classifica- 1999; Gómez-Zurita et al. 2000, Durando et al. tion of the family Tephritidae, based primarily on 2000), is particularly useful. morphological data, is still a matter of debate A molecular study was therefore undertaken to (White 1996; Korneyev 2001). Many characters compare a DNA region of the mitochondrial are difficult to interpret, and disagreements in cytochrome oxidase II gene from different tephritid the definition of groups have arisen owing to the species belonging to six different genera, in or- relative importance that researchers place on der to obtain an estimate of the phylogenetic rela- these characters (Drew 1989). tionships within some members of this dipteran In the last two decades the development of family. molecular techniques, based mostly on PCR and/ or sequencing, has provided new and powerful MATERIALS AND METHODS tools to address the most diverse of biological problems. Taxonomy has profited much from Samples and DNA isolation them. A more accurate classification of the Insect samples included 16 species representing Tephritidae might be gained through the use of six genera in the family Tephritidae, namely, molecular techniques. Indeed, a number of recent Ceratitis, Rhagoletis, Dacus, Bactrocera, Anastrepha studies using molecular data have contributed to and Toxotrypana, from representative areas of each taxon distribution (Table 1). *To whom correspondence should be addressed. E-mail: [email protected] Total DNA from individual adults or pupae was 440 Proceedings of the 6th International Fruit Fly Symposium Table 1. Species used and origin of the samples. were analysed using an ABIPRISM 377 DNA sequencer (Applied Biosystems). Species Origin Sequence alignment and phylogenetic analysis Anastrepha obliqua Mexico Alignment of the sequences and the estimation Anastrepha striata Mexico of interspecific pairwise percentage sequence Anastrepha suspensa U.S.A. (Florida) divergence was performed by the clustal method Bactrocera cacuminata Australia using the MegAlign programme included in the Bactrocera cucurbitae U.S.A. (Hawaii) DNASTAR package (Lasergene System 1994). Bactrocera dorsalis U.S.A. (Hawaii) A neighbour-joining (NJ) tree (Saitou & Nei Bactrocera latifrons U.S.A. (Hawaii) 1987) was generated from the Jukes-Cantor Bactrocera oleae Spain distances (Jukes & Cantor 1969) using Drosophila Bactrocera zonata Mauritius Island yakuba and Liriomyza huidobrensis as outgroups Ceratitis capitata Spain (accession numbers 12918 and AF327292, Gene- Ceratitis cosyra South Africa Bank database). Dacus ciliatus Réunion Island Dacus demmerezi Réunion Island The reliability of the tree was evaluated using Rhagoletis cerasi Switzerland 1000 bootstrap replicates (Felsenstein 1985), Rhagoletis pomonella U.S.A. (New York) and by constructing a consensus tree from the Toxotrypana curvicauda U.S.A. (Florida) 1000 bootstrapped trees obtained. Phylogenetic analysis was performed using the PHYLIP software package (Felsenstein 1993). isolated applying a method specific for small samples, using SDS dissolution, phenol-chloro- RESULTS AND DISCUSSION form extraction and ethanol precipitation (Reyes Nucleotide sequences of 259 bp were obtained et al. 1997). for the cytochrome oxidase II region studied. Interspecific pairwise percentage sequence DNA amplification and sequencing differences ranged from 0.8 to 22.8% (Table 2), The polymerase chain reaction (PCR) was used to with a mean of 16%. The lowest value observed amplify a 259 bp segment of the mitochondrial was for comparisons between Bactrocera dorsalis cytochrome oxidase II gene. The primers used were and Bactrocera cacuminata (0.8%). The highest CO2A (5’-GGACTACAAGATAGAGCCTC-3’) and interspecific divergence was found between CO2B (5’-CTTCAGTATCATTGATGACC-3’), provided Anastrepha suspensa and Bactrocera latifrons by C. Fleming (The Queen’s University of Belfast, (22.8%). The mean of 16% for the interspecific U.K.). The amplified fragment corresponds to posi- sequence differences is close to the 11% found tions 3124-3382 of the mitochondrial genome of by Han & McPheron (1997) in representative Drosophila yakuba (Clary & Wolstenholme 1985). species of the family Tephritidae (based on the Amplifications were performed in a PTC-100 analysis of a fragment of mitochondrial 16S MJ research thermocycler, in 100 µl reactions ribosomal DNA). The sequence divergence values containing 1 µl of genomic DNA, 1 µM of each obtained in the present work were generally low primer, 0.2 mM of each dNTP,2 mM of MgCl2,10µl (20% or less). Comparisons of these results with of 10 × Eco Taq buffer (Ecogen), and 2.5 units of those reported by others authors show that Eco Taq polymerase (Ecogen). PCR conditions in- they would normally be thought to correspond to volved a initial cycle of 5 min at 94°C, 30 cycles of related species. In the case of Bactrocera dorsalis 30 s at 94°C, 1 min at 55°C or 57°C (Bactrocera and Bactrocera cacuminata, a divergence of dorsalis), and 1 min 30 s at 72°C. The final cycle 0.8% falls in the range of values generally found had an additional step of 6 min at 72°C. Nega- at the intraspecific levels. This close relationship tive controls were always included. PCR prod- between these species agrees with recent classifi- ucts were purified using a PCR purification kit cations which place them both in the ‘Bactrocera (Boehringer). dorsalis complex’ (White & Elson-Harris 1994). Nucleotide sequences were determined directly This is a group of morphologically inseparable but from PCR fragments using the dideoxy chain biologically distinct species whose members may termination method (Sanger et al. 1977) with be referred to as sibling species (White 1996). fluorescently-labelled dideoxynucleotide termina- In the genus Rhagoletis, comparisons among tors (Applied Biosystems). Sequencing reactions members of the pomonella and cingulata groups Fernandez et al.: A phylogenetic study of the family Tephritidae using mitochondrial DNA 441 Table 2. Percentage sequence divergence in the cytochrome oxidase II gene between tephritid species. A. obliqua * A. suspensa 1.5 * A. striata 5.8 6.2 * B. cucurbitae 18.1 17.8 18.5 * B. cacuminata 18.9 19.7 17.4 13.9 * B. dorsalis 18.1 18.9 17.4 13.9 0.8 * B. latifrons 22.4 22.8 19.7 16.6 13.9 13.5 * B. oleae 19.3 19.3 20.1 16.6 15.4 15.8 17.0 * B. zonata 18.9 19.3 18.1 12.4 6.9 6.6 11.2 15.1 * C. cosyra 15.1 15.1 17.0 16.6 15.8 15.4 19.7 18.9 16.6 * C. capitata 13.1 13.1 14.7 17.4 15.4 15.1 20.8 17.4 16.6 6.6 * D. ciliatus 14.3 13.5 15.1 14.7 18.1 17.4 18.5 17.0 15.4 17.0 15.1 * D. demmerezi 18.1 18.9 19.7 16.6 14.3 13.9 16.2 16.2 15.8 16.6 17.0 10.8 * R. cerasi 16.6 16.2 15.8 18.1 17.0 17.4 21.2 21.2 18.9 18.5 17.0 17.4 20.5 * R. pomonella 13.5 13.9 15.4 17.0 15.8 15.4 19.7 17.4 16.6 15.1 11.6 15.1 17.8 14.3 * T. curvicauda 14.3 14.7 13.5 18.1 15.8 15.8 20.8 19.3 18.1 13.1 13.1 19.3 17.8 18.1 15.8 * 123456789 10111213141516 showed sequence divergences below 0.5% species of the genera Dacus, Bactrocera and (McPheron & Han 1997).