1. 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 Tephritid Barcoding Initiative and barcoding of agricultural pest

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

3. Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences The Tephritid Barcode Initiative (TBI) TBI Chair: Bruce McPheron, Penn State TBI Coordinators: Allen Norrbom, USDA, USA Marc De Meyer, RMCA, Belgium 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

4. Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences 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

5. Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences 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

6. Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences Who is generating fruit fly barcodes? 1.Penn State University, USA: Bruce McPheron, Md. Sajedul Islam 2.Lincoln University, New Zealand: Karen Armstrong 3.Royal Museum Central Africa, BE: Marc De Meyer, Massi Virgilio 4.Yonsei University, Korea: Ho-Yeon Han 5.California Department of Agriculture, USA: Peter Kerr 6.Smithsonian National Museum of Natural History, USA: Allen Norrbom 7.APHIS-PPQ Mission lab, USA: Norman Barr 8.University of Guelph 9.Biodiversity Institute of Ontario

7. Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences 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

8. Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences What needs to be provided in BOLD for TBI Euleia fratria (Trypetinae)TEPH101 (from BOLD)Trypetinae 1.identification of specimen by an expert taxonomist 2.voucher specimen 3.collection information (collection date and location) 4.other infos (GPS, elevation, photodocumentation) not mandatory but strongly encouraged 5.barcode: at least 500bp with less than 1% missing data. 6.trace files stored in BOLD. Other COI records (e.g., Genbank submissions) are integrated into the BOLD database but kept separate.

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

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

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

12. 0% 4% 8% 12% 16% 20% BactroceraCeratitisDacus between species within species intra- and inter-specific genetic differentiation in 3 genera of tephritid fruitflies p-distances 12.6 6.3 6.6 ratio inter/intra specific p-dist 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 K2p = 0.02% (S.E.= 0.01%) average genetic distance within species

14. Ceratitis fasciventris C. anonae C. rosa... and where not: the FAR complex 0.00 0.01 0.02 within species between species K2P average genetic distances 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 taxonomically closer species... D. chiwira - D. famona, p-dist.= 0.3% (subgenus Dacus) D. apostata - D. triater, p-dist.= 0.0% (subgenus Psilodacus)

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

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

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

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

21. Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences 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. methodological problems in the barcoding of museum specimens

22. age of specimens vs barcoding success % specimens amplified % specimens sequenced methodological problems in the barcoding of museum specimens 0 20 40 60 80 100 20072006200520042003200220012000 90s80s < 1980 (n=394)

23. 0 20 40 60 80 100 20072006200520042003200220012000 90s80s < 1980 from EtOH specimens from pinned specimens % of succesfully sequenced specimens (n=394) methodological problems in the barcoding of museum specimens pinned vs EtOH preserved specimens

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

25. 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. methodological problems in the barcoding of museum specimens Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

26. 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 methodological problems in the barcoding of museum specimens

27. Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences 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 methodological problems in the barcoding of museum specimens

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. % of pcr products obtained % of barcode sequences obtained (>500bp)

30. higher performances compared to the standard primers 0 20 40 60 80 100 >200090s80s<1980 standard primers internal primers (n=229) +7%+32%+6%+7% a new set of internal primers for the barcoding of tephritids % of barcodes obtained (>500bp)

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

32. work in progress: could ‘minibarcodes’ represent a temporary alternative to ‘full’ barcodes?

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

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

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

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 to collaborations 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. Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences 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)