presented by M. Virgilio Royal Museum for Central Africa, Tervuren, Belgium

1. Tephritid Barcoding Initiative and barcoding of agricultural pest presented by M. Virgilio Royal Museum for Central Africa, Tervuren, Belgium Royal Belgian Institute of Natural Sciences, Brussels, Belgium m.virgilio@skynet.be Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

2. Why DNA Barcode Fruit Flies? • large number of species • many economically relevant species • difficult identification of larvae. • need for standardized diagnostic methods Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

3. The Tephritid Barcode Initiative (TBI) • CBOL obtained funding from the Sloan Foundation • to support a “Demonstrator System” • Steering Committee formed in April, 2006, in Belgium Steering Committee Members: Karen Armstrong, New Zealand Norman Barr, USA Amnon Freidberg, Israel Ho-Yeon Han, South Korea George Roderick, USA Ian White, UK TBI Chair: Bruce McPheron, Penn State TBI Coordinators: Allen Norrbom, USDA, USA Marc De Meyer, RMCA, Belgium Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

4. TBI proposal: Goals Generate barcode database for 2,000 species • 5 individuals/species (10,000 specimens) • 100% of economically important (EI) species • >75% of EI congeners • 1 species per genus in subtribes containing EI species • 1 species per genus in other higher taxa • representative tephritoid families Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

5. TBI proposal: Beneficial Outcomes 1) Establish a globally-available DNA database of barcodes 2) Establish a globally-available DNA repository 3) Generate a collection of identified vouchers for future systematic work Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

6. Who is generating fruit fly barcodes? • Penn State University, USA: Bruce McPheron, Md. Sajedul Islam • Lincoln University, New Zealand: Karen Armstrong • Royal Museum Central Africa, BE: Marc De Meyer, Massi Virgilio • Yonsei University, Korea: Ho-Yeon Han • California Department of Agriculture, USA: Peter Kerr • Smithsonian National Museum of Natural History, USA: Allen Norrbom • APHIS-PPQ Mission lab, USA: Norman Barr • University of Guelph • Biodiversity Institute of Ontario Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

7. Financial support Penn State University, USA: technical support APHIS: collecting & sequencing Belgian Federal Government: sequencing, pilot study mini-barcodes California Citrus Board: general expenditure Korean Government: sequencing (Dutch Government: collecting) Various contributions through staff time and general core funding of institutions or other projects Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

8. What needs to be provided in BOLD for TBI • identification of specimen by an expert taxonomist • voucher specimen • collection information (collection date and location) • other infos (GPS, elevation, photodocumentation) not mandatorybut strongly encouraged • barcode: at least 500bp with less than 1% missing data. • trace files stored in BOLD. Euleia fratria (Trypetinae)TEPH101 (from BOLD) Other COI records (e.g., Genbank submissions) are integrated into the BOLD database but kept separate. Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

9. http://www.barcodinglife.org Tephritidae {family} Barcodes : Species : Search performed on October 20, 2008 Lineage: Arthropoda: Insecta: Diptera Specimen Record: 1047 Specimens with barcodes: 828 Public Sequences: 240 Subfamilies (5) Dacinae [572] Phytalmiinae [7] Tachiniscinae [1] Tephritinae [219] Trypetinae [243] Deposited in :

10. all species EI species % of species barcoded 100 80 60 40 20 0 Dacus Ceratitis Rhagoletis Bactrocera Anastrepha Five Genera of Highest Economic Importance number of species 600 533 400 240 213 200 119 95 73 44 37 34 19 0 Dacus Ceratitis Rhagoletis Bactrocera Anastrepha

11. insights into DNA barcoding of tephritid fruitflies Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

12. intra- and inter-specific genetic differentiation in 3 genera of tephritid fruitflies ratio inter/intra specific p-dist 12.6 6.3 6.6 20% 16% p-distances 12% between species 8% within species 4% 0% Bactrocera Ceratitis Dacus Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

13. DNA barcoding of tephritid fruitflies: where it works nicely.... 44 barcodes of Bactrocera cucurbitae from 11 countries: • Bangladesh • Cambodia • China • Hawaii • India • Kenya • La Reunion • Malaysia • Philippines • Sudan • Tanzania average genetic distance within species K2p = 0.02% (S.E.= 0.01%)

14. ... and where not: the FAR complex Ceratitis fasciventris C. anonae C. rosa average genetic distances 0.02 K2P 0.01 between species within species 0.00 ratio = 1.2

15. other potentially “problematic” tephritid species groups and complexes: B. dorsalis complex C. cosyra complex C. capitata / C. caetrata T. occipitale / T. quadrimaculatum ... Armstrong and Ball (2005) Phil. Trans. R. Soc. B 360: 1813-1823. (B. dorsalis complex) Virgilio et al. (2008) Molecular Phylogenetic and Evolution 48: 270-280 (FAR complex, Ceratitis)

16. Dacus: from identification issues to classification issues as expected low genetic differentiation in taxonomicallycloserspecies... D. chiwira - D. famona, p-dist.= 0.3%(subgenus Dacus) D. apostata - D. triater, p-dist.= 0.0% (subgenus Psilodacus)

17. DACUS DIDACUS LEPTOXYDA LOPHODACUS NEODACUS PSILODACUS COI NJT-K2P ...but also discrepancies between morphological and molecular taxonomy subgeneric classification

18. Apocynaceae Cucurbitaceae Passifloraceae unknown DNA barcoding may provide clues for an improved morphological classification host plant choice COI+16S+per Bayesian

19. DNA barcoding may reveal cryptic speciation: Virgilio M., De Meyer M, WhiteI.M., Backeljau T. (submitted) Phylogenetic relationships among African Dacus species (Diptera: Tephritidae) as inferred from mitochondrial and nuclear DNA.

20. methodological problems in the barcoding of tephritid fruitflies from museum collections Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

21. methodological problems in the barcoding of museum specimens DNA Extraction Procedures: Several pilot studies performed to assess the success of an invasive, nondestructive DNA extraction method for museum specimens. Study 1: L. Weigt and A. Driskell (Laboratory of Analytical Biology, National Museum of Natural History, Smithsonian Institution) -16S and COI Study 2: N. Barr (USDA) and R. Ruiz (Penn State) -16S Study 3. M. Virgilio (Royal Museum Central Africa, BE) – 16S, COI Study 4. Md. Sajedul Islam (Penn State) - COI (ongoing study) Qiagen DNeasy kit: ok for DNA from recent dry and alcohol specimens not crushed during the extraction process. Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

22. 100 % specimens amplified 80 % specimens sequenced 60 40 20 0 90s 80s 2007 2006 2005 2004 2003 2002 2001 2000 < 1980 methodological problems in the barcoding of museum specimens age of specimens vs barcoding success (n=394)

23. 100 from EtOH specimens 80 from pinned specimens 60 40 20 0 < 1980 2007 2006 2005 2004 2003 2002 2001 2000 90s 80s methodological problems in the barcoding of museum specimens pinned vs EtOH preserved specimens % of succesfully sequenced specimens (n=394)

24. <1940 >2000 32% 41% 40s 4% 50s 90s 80s 13% 70s 4% 3% 1% 60s 2% methodological problems in the barcoding of museum specimens Ceratitis, Bactrocera and Dacus in the collections of the RMCA: (n=1804)

25. methodological problems in the barcoding of museum specimens DNA extraction protocols tested: • Qiagen DNeasy • Qiagen Biosprint • Bio101 Ancient DNA kit • Autogen • Machery-Nagel Filterservice kit • Phenol-Chloroform • DNAzol • E.Z.N.A. kits for forensics and insects • chargeswitch magnetic beads • etc. Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

26. methodological problems in the barcoding of museum specimens • problem: • the quality of DNA rapidly decreases in time (shearing) • the amount of barcodes obtained from older museum specimens is not significantly affected by • the extraction method used • the use of genus- and species-specific primers • objective: • develop internal primers to improve the collection of barcodes from sheared DNA Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

27. methodological problems in the barcoding of museum specimens • the BARFLY project • short term project funded by the Belgian Science Policy (BELSPO) • Joint Experimental Molecular Unit • of RMCA and RBINS (www.jemu.be) • Jeroen Van Houdt,Floris Breman • development of internal primers for the barcoding of Tephritids • collection of new barcodes from museum specimens Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

28. LCO 1490 HCO 2198 full barcode - c. 670 bp frag. 1 - 343bp frag. 2 - 269bp frag. 3 - 227bp a new set of internal primers for the barcoding of tephritids VanHoudt J., Breman F. C., Virgillio M.,De Meyer M. (in prep.) A protocol for DNA barcoding of African tephritid fruitflies from museum collections using mini barcodes.

29. 100 (n=229) 80 60 40 fragment 1 20 fragment 2 0 >2000 90s 80s 70s 60s 50s 40s <1940 fragment 3 100 . (n=229) 80 60 40 20 0 >2000 90s 80s 70s 60s 50s 40s <1940 % of pcr products obtained % of barcode sequences obtained (>500bp)

30. +7% +32% +6% +7% 100 80 60 standard primers 40 internal primers 20 (n=229) 0 >2000 90s 80s <1980 a new set of internal primers for the barcoding of tephritids higher performances compared to the standard primers % of barcodes obtained (>500bp)

31. a new set of internal primers for the barcoding of tephritids RMCA: internal primers as a standard protocol for the barcoding of museum specimens collected before 2000 Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

32. work in progress: could ‘minibarcodes’represent a temporary alternative to ‘full’ barcodes? Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

33. Dacus armatus 400 bp 400 bp 500 bp 600 bp 400 bp Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

34. Bactrocera oleae 500 bp 300 bp 400 bp Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

35. TBI current challenges • Coordination / tracking specimens: BOLD • Older specimens: mini barcodes / internal primers • Fresh material: collecting activities • Taxon coverage: non EI genera Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

36. RMCA current challenges RMCA is collecting barcodes as a “complementary activity” to the currently ongoing research lines: • phylogeny and population genetics of African Dacus • molecular taxonomy of species complexes in the genus Ceratitis • phylogeny of African and Australasian Bactrocera • population genetics and phylogeography of B. cucurbitae Marc De Meyer: marc.de.meyer@africamuseum.be Massimiliano Virgilio: m.virgilio@skynet.be contact:

37. RMCA current challenges RMCA is opened tocollaborations with African Institutions aiming to: • identify tephritid samples through morphological and molecular characters • prepare species inventories from African countries • collect / rear tephritid fruit flies • investigate population genetics and host races evolution of tephritid species Marc De Meyer: marc.de.meyer@africamuseum.be Massimiliano Virgilio: m.virgilio@skynet.be contact:

38. Acknowledgments Karen Armstrong Abdelaziz Babikir Thierry Backeljau Norman Barr Marc DeMeyer Sajedul Islam Bruce McPheron researches at the RMCA are currently funded by the Belgian Science Policy Action 1 (project MO/37/017) and benefit from practical support by the Joint Experimental Molecular Unit (JEMU) Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences