This paper marks the first attempt at using the molecular clock analysis on five Malaysian Bactrocera species based on combined data of COI and ND1 to test the divergence times of species that infested 11 varieties of fruits and vegetables. A divergence tree was constructed to estimate the cryptic species among the five closely related Bactrocera species viz. B. (Bactrocera) and B. (Zeugodacus). Both subgenera evolved and diverged approximately 21.5933 million years ago (MYA). The particularly complex taxonomical status of the sister-species B. papayae and B. carambolae has been resolved using the divergence tree; the two separated from the others approximately 2.743 MYA, while B. papayae and B. carambolae distinctly diverged between 1.9619 and 2.2884 MYA. Additionally, Bactrocera dorsalis showed divergence at 4.1596 MYA and was distinctly separate from the B. papayae. Interestingly, the molecular clock results are congruent with the phylogenetic tree that was analysed using Maximum Parsimony and Bayesian Inference. Further, the taxonomical status and host utilisation of the five Bactrocera species were also briefly discussed, which indicated that B. cucurbitae (subgenus Zeugodacus) was infesting Cucurbitaceae and B. latifrons was infesting Solanaceaea, while B. dorsalis, B. carambolae and B. papaya were infesting a wide range of fruit species. Key message Divergence time of five Malaysian Bactroceran species is reported for the first time and found congruent with the results of phylogeny based on combined data of COI and ND1 markers using MP and Bayesian analyses. This finding could prove very crucial for resolving the most crucial taxonomic conflicts in Bactrocera classification, which is highly important initially in species identification for integrated pest management (IPM) programme.

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Malaysia is a tropical country that produces commercial fruits, including star fruits, Averrhoa carambola L. (Oxalidales: Oxalidaceae), and guavas, Psidium guajava L. (Myrtales: Myrtaceae). There is a high demand for these fruits, and they are planted for both local consumption and export purposes. Unfortunately, there has been a gradual reduction of these fruits, which has been shown to be related to fruit fly infestation, especially from the Bactrocera species. Most parasitic wasps (Hymenoptera: Braconidae: Opiinae) are known as parasitoids of fruit fly larvae. In this study, star fruits and guavas infested by fruit fry larvae were collected from the Malaysian Agricultural Research and Development Institute. The parasitized larvae were reared under laboratory conditions until the emergence of adult parasitoids. Multiplex PCR was performed to determine the braconid species using two mitochondrial DNA markers, namely cytochrome oxidase subunit I and cytochrome b . Two benefits of using multiplex PCR are the targeted bands can be amplified simultaneously using the same reaction and the identification process of the braconid species can be done accurately and rapidly. The species of fruit flies were confirmed using the COI marker. The results obtained from our study show that Diachasmimorpha longicaudata (Ashmead) (Hymenoptera: Braconidae), Fopius arisanus (Sonan), and Pysttalia incisi (Silvestri) were parasitoids associated with Bactrocera carambolae (Drew and Hancock) (Diptera: Tephritidae) infested star fruits. Fopius arisanus was also the parasitoid associated with Bactrocera papayae (Drew and Hancock) infested guavas. Maximum parsimony was been constructed in Opiinae species to compare tree resolution between these two genes in differentiating among closely related species. The confirmation of the relationship between braconids and fruit fly species is very important, recognized as preliminary data, and highly necessary in biological control programs.

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