1. Research Projects Hawaiian Drosophilidae –Taxonomy –Molecular systematics –Rates of diversification –Molecular evolution –Ecology

2. Research Projects Hawaiian Drosophilidae –Taxonomy –Molecular systematics –Rates of diversification –Molecular evolution –Ecology Drosophila repleta species group –Population structure –Molecular systematics –Ecology

3. Research Projects Hawaiian Drosophilidae –Taxonomy –Molecular systematics –Rates of diversification –Molecular evolution –Ecology Drosophila repleta species group –Population structure –Molecular systematics –Ecology Family Drosophilidae –Genus-level relationships molecular and morphological data –Various phylogenetic studies saltans and obscura groups subgenus Sophophora

4. Problems and prospects in drosophilid systematics…. Problems: –Incongruence between morphological and molecular characters

5. Morphology Stegana Rhinoleucophenga Amiota Gitona Leucophenga Scaptodrosophila Chymomyza Paramycodrosophila Hirtodrosophila Zygothrica Mycodrosophila Hawaiian Drosophila Engiscaptomyza Continental Scaptomyza Hawaiian Scaptomyza Drosophila Sophophora Zaprionus Samoaia Liodrosophila after Grimaldi (1990)

6. Morphology Stegana Rhinoleucophenga Amiota Gitona Leucophenga Scaptodrosophila Chymomyza Paramycodrosophila Hirtodrosophila Zygothrica Mycodrosophila Hawaiian Drosophila Engiscaptomyza Continental Scaptomyza Hawaiian Scaptomyza Drosophila Sophophora Zaprionus Samoaia Liodrosophila vs. Molecules after Grimaldi (1990) after Remsen & DeSalle (1998)

7. Reasons for conflict: –different methods of analysis –more extensive taxon sampling in the morphological study

8. Reasons for conflict: –different methods of analysis –more extensive taxon sampling in the morphological study Goals: –standardize analytical methods and combine data –increase taxon sampling in molecular data Examined approximately 60 clades (genera, subgenera, and species groups) of drosophilid flies. About 1/3 of these were from field collections. 3X the number of genera previously studied. Examined over half of the taxonomically defined subgenera in the genus Drosophila. Included roughly half of the ~ 50 species groups in the subgenus Drosophila.

9. Problems and prospects in drosophilid systematics…. Problems: –Incongruence between morphological and molecular characters –Poor support at some nodes in combined analyses (~3kb of data per taxon)

10. * * * * * * * * * * Problems: - Bootstrap support at several key nodes in the tree is weak.

11. * * * * * * * * * * Problems: - Bootstrap support at several key nodes in the tree is weak. - May be the result of a rapid adaptive radiation at the base of Drosophilinae (~50-60 MYA).

12. * * * * * * * * * * Problems: - Bootstrap support at several key nodes in the tree is weak. - May be the result of a rapid adaptive radiation at the base of Drosophilinae (~50-60 MYA). - More characters will be needed to resolve these relationships.

13. Problems and prospects in drosophilid systematics…. Problems: –Incongruence between morphological and molecular characters –Poor support at some nodes in combined analyses (~3kb of data per taxon) Prospects: –High throughput sequencing methods and a “shallow genomic” approach to character sampling.

14. Project Goals Generate a phylogenetic hypothesis for relationships in Drosophilidae using a large number of gene sequences and taxa.

15. Project Goals Generate a phylogenetic hypothesis for relationships in Drosophilidae using a large number of gene sequences and taxa. –The stated goal is 56 loci for about 120 taxa.

16. Project Goals Generate a phylogenetic hypothesis for relationships in Drosophilidae using a large number of gene sequences and taxa. –The stated goal is 56 loci for about 120 taxa. –Additional loci and taxa will be added, if possible

17. Project Goals Examine molecular evolution of these loci to better understand a variety of phenomena, including genome evolution.

18. Project Goals Examine molecular evolution of these loci to better understand a variety of phenomena, including genome evolution. Study character interaction among gene partitions.

19. Project Goals Examine molecular evolution of these loci to better understand a variety of phenomena, including genome evolution. Study character interaction among gene partitions. What are the best analytical methods given the size of this data set?

20. Project Goals Examine molecular evolution of these loci to better understand a variety of phenomena, including genome evolution. Study character interaction among gene partitions. What are the best analytical methods given the size of this data set? How do you voucher, database, annotate, and archive such a large amount of data?

21. Methods High throughput approach –Use 96 well plates to organize taxa and primers.

22. Methods High throughput approach –Use 96 well plates to organize taxa and primers. A Primer plates: 12 columns across will contain pre- mixed solutions of 12 primer sets (A-L). These will be stored in large enough volumes such that 20 reactions can be done using a single plate. - Column “A” contains 20X of primers Aright and Aleft.

23. Methods High throughput approach –Use 96 well plates to organize taxa and primers. A Primer plates: 12 columns across will contain pre- mixed solutions of 12 primer sets (A-L). These will be stored in large enough volumes such that 20 reactions can be done using a single plate. - Column “A” contains 20X of primers Aright and Aleft. 1 Taxa plates: 8 rows down will contain genomic DNA from the taxa we wish to sample (1-8). DNA will be quantified and stored in working concentrations. - Row “1” contains enough DNA to perform 20 PCR reactions.

24. A 1 PCR: Pipette an aliquot of each primer pair and each taxon into a plate containing PCR beads, add Taq with repeater pipette, and place in PCR machine. + 1 A + Taq Taxa and primer plates can be mixed and matched so that all taxa can be determined for all loci.

25. Primers Five primer plates will be needed to sequence the 56 loci we have identified for this study. Plate 1: esc, sia, snf, amy, glass, fkh, boss, snf, ntid, kuz, cg3861, cg2864 Plate 2: Sod, Adh, 28Sa, 28Sb, Gpdh, amd, COII, Yp1, Yp2, 16S, ef1a, COI Plate 3: bicoid, wee, cg7036, ovo, fzr, cg6121, mad, hep (and four others under development) Plate 4: all mitochondrial loci Plate 5: 12 nuclear genes under development Additional loci will be added as needed.

26. More Primers Primer design and testing continues…. Close to 100 primers will be in use by the end of year one.

27. Taxa 8 species in the lab will be tested in plates over the next two months. 45 species will be ordered from the Drosophila Species Stock Center in Tucson. They will be available for sequencing in early February. Field collections in Mexico, Costa Rica, and Hawai‘i are planned for spring-summer 2002. –Hawai‘i and Mexico trips should produce a total of 25 species for sequencing. –Costa Rica trip will yield between 30-40 taxa, most of which can be used for sequencing. Total of about 110 taxa by the end of year one.

28. More Taxa Additional species will be ordered from Tucson Stock Center in year two. Collecting trips to Vietnam and Brasil will take place in year two. Total project should approach 200 taxa.

29. Purification of PCR products, sequencing reactions, and clean up of sequencing reactions will continue in the 96 well plate format. –Sequencing reactions will require two plates, one with each primer. The robot will be used to further automate this process.

30. Notes on data management This is a complicated project linking the research efforts of several researchers –Rob, Patrick, Bonacum - genus level Drosophilidae phylogeny –Deodoro - repleta phylogeny –Julian - antopocerus phylogeny

31. Notes on data management This is a complicated project linking the research efforts of several researchers –Rob, Patrick, Bonacum - genus level Drosophilidae phylogeny –Deodoro - repleta phylogeny –Julian - antopocerus phylogeny Data handling must be standardized. –Sequencher will be used to organize all gene sequences –Standard NEXUS format will be adopted

32. Notes on data management This is a complicated project linking the research efforts of several researchers –Rob, Patrick, Bonacum - genus level Drosophilidae phylogeny –Deodoro - repleta phylogeny –Julian - antopocerus phylogeny Data handling must be standardized. –Sequencher will be used to organize all gene sequences –Standard NEXUS format will be adopted All taxa in this study have FTC accession numbers, linking them not only to material in frozen tissue, but to voucher material in the AMNH pinned collection. Every step, from DNA isolation and quantification to sequence alignment, will use this number.

33. Notes on data management PDAs will be used, in conjunction with mobile database software, as electronic lab notebooks. All lab procedures will be recorded, synched with a server machine and archived in the database.

34. Notes on data management PDAs will be used, in conjunction with mobile database software, as electronic lab notebooks. All lab procedures will be recorded, synched with a server machine and archived in the database. Another PDA will be used as a field notebook.

35. Notes on data management PDAs will be used, in conjunction with mobile database software, as electronic lab notebooks. All lab procedures will be recorded, synched with a server machine and archived in the database. Another PDA will be used as a field notebook. Server machine will –host project website - including searchable online databases for primers, published sequences, and vouchers –archive all relevant data