Barcoding Type Specimens

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

2. Outline • Ancient DNA • Project overview • Purpose • Primary goals • Obstacles and solutions • Current progress • Preliminary findings • 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 • Ancient DNA • Project overview • Purpose • Primary goals • Obstacles and solutions • Current progress • Preliminary findings • Future prospects

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

7. Primary goals • Develop type specimen protocol • Effective, cheap, high-throughput • Apply protocol to: • ~3000 geometrids • ~300 xyloryctids • Other Lepidoptera families • 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 • Some legs <10 ug • 100,000 fold less template than standard ancient bone samples • Variable killing and storage conditions • Variable handling over the decades 400 mg 0.04 mg

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

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

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

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

14. Outline • Ancient DNA • Project overview • Purpose • Primary goals • Obstacles and solutions • Current progress • Preliminary findings • Future prospects

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

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

17. Quality of Data

18. Outline • Ancient DNA • Project overview • Purpose • Primary goals • Obstacles and solutions • Current progress • Preliminary findings • Future prospects

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

20. Does age affect success?

21. Does size affect success?

22. Outline • Ancient DNA • Project overview • Purpose • Primary goals • Obstacles and solutions • Current progress • Preliminary findings • 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 Human Contaminants REAL Primer Dimers

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

26. Acknowledgments Funding provided by: