1. Barcoding Type Specimens Goals, Obstacles, and Current Progress

2. Outline 1.Ancient DNA 2.Project overview a. Purpose b. Primary goals c. Obstacles and solutions 3.Current progress 4.Preliminary findings 5.Future prospects

3. A primer on ancient DNA >100 years old is considered "ancient” Quality (%) Age(yrs)

4. How old can we go?

5. Outline 1.Ancient DNA 2.Project overview a. Purpose b. Primary goals c. Obstacles and solutions 3.Current progress 4.Preliminary findings 5.Future prospects

6. Why barcode type specimens? Barcode database construction o Helps put names to faces Establish links to modern specimens o Taxonomic clarification o Help resolve cryptic species complexes o Help resolve unnecessary splits

7. Primary goals Develop type specimen protocol o Effective, cheap, high-throughput Apply protocol to: o ~3000 geometrids o ~300 xyloryctids o Other Lepidoptera families o Other orders

8. Oldest legitimate DNA? 130,000 years old 45,000 years old 700,000 years old

9. Obstacles Small tissue size = less DNA o Some legs <10 ug o 100,000 fold less template than standard ancient bone samples Variable killing and storage conditions Variable handling over the decades 400 mg0.04 mg

10. Solutions: Tissue size Abdominal lysates o Alternative to legs o Made prior to dissections o More DNA than a single leg Concentrate DNA extract Increase PCR cycles

11. Additional obstacles Project specific o Hundreds of species  Universal primers o Contaminants  Universal primers may also amplify contaminants  Cannot wash or take "core" sample from tissue o High-throughput  Must be cost effective  Cannot extensively focus on any one sample

12. Solutions: Many species Universal primers o Degenerate primers o Primer cocktail Target conserved yet hypervariable region o 164 bp region of COI Second attempt for specimens that failed first pass o 94 bp region of COI 658 bp BARCODE 164 bp 94 bp

13. Solutions: Contamination Dedicated room, equipment, reagents, and workstations Sterile practices o Full body suit, hood, mask, gloves o Frequent glove changes o Avoid working over samples o Avoid generating aerosols

14. Outline 1.Ancient DNA 2.Project overview a. Purpose b. Primary goals c. Obstacles and solutions 3.Current progress 4.Preliminary findings 5.Future prospects

15. Type Specimen Protocol LYSIS EXTRACTION DNA (non-destructive)(single column) PCR SEQUENCING EDITING (164 bp) (94 bp) (658 bp)

16. Current Progress Processed: o Geometridae: 948/3000 (32% complete) o Xyloryctidae: 224/300 (75% complete) o Other: 383/383 (100% complete) o Total:1555/3683 (42% complete) Success: o Geometridae: 629/948 = 66% o Xyloryctidae: 103/224 = 46% o Other: 278/383 = 73% o Total: 1010/1555 = 65%

17. Quality of Data

18. Outline 1.Ancient DNA 2.Project overview a. Purpose b. Primary goals c. Obstacles and solutions 3.Current progress 4.Preliminary findings 5.Future prospects

19. Factors affecting success Taxonomy o Tissue size o Primer binding efficiency Age Killing method Handling Storage

20. Does age affect success?

21. Does size affect success?

22. Outline 1.Ancient DNA 2.Project overview a. Purpose b. Primary goals c. Obstacles and solutions 3.Current progress 4.Preliminary findings 5.Future prospects

23. Destructive processing Pre-lysis tissue grinding Applicable to dry legs Hypothesis: Grinding tissue prior to lysis will increase accessibility of DNA

24. Real-time PCR Monitor amplification in real-time Sequence ONLY true positives REAL Primer Dimers Human Contaminants

25. Further Experiments Samples: Ancient lep specimens we can destroy Allows us to measure one variable while controlling all others Test: o Pre-lysis tissue treatments o Tissue types o Size (i.e. mass) o Primer binding efficiency

26. Acknowledgments Funding provided by: