1. Policies and best practices for molecular analysis of museum specimens Peter T Oboyski Essig Museum of Entomology UC Berkeley

2. “All of this has happened before, and it will all happen again.” ECN meeting 1995 – Jim Whitfield

3. What molecular analyses? DNA sequencing of the specimen Parasites, pathogens, or pollen Stable isotope analyses

4. The Problem With the advances in molecular techniques and molecular systematics we can expect to see more loan requests for molecular analyses – How to respond? Destructive sampling is in conflict with preservation – At the discretion of the curator – Recent methods do not require grinding tissues Policies need to be in place to respond to requests

5. Policy Recommendations I: Necessity Is there just cause to use museum specimens? – Historical analyses – “Impossible” to access collecting locations – Local extirpation or extinct species Xerces blue

9. Necessity: Availability of specimens Long series are no problem – What about types or rare specimens?

10. British Museum microlepidoptera types – New policy to extract DNA during genitalia preps

11. Policy Recommendations II: Methods Does the researcher have the proper facilities and methods to likely recover DNA? – “Clean room” or “Ancient DNA” room – Mitochondrial vs. Nuclear DNA – Fragment length of gene region to sequence Single Nucleotide Polymorphisms (SNPs) – Sanger sequencing vs. Next Generation – Extraction method Phenol-chloroform extractions vs. Qiagen ® kits

12. Policy Recommendations III: Qualifications Does the researcher have the proper background and experience for success? – Experiment with other specimens (not mine!) – Letter of reference from advisor or DNA facility – Read between the lines of the proposal/methods

13. Other considerations Scientific merit of the project Specimen history: kill method, drying, storage Specimen identification: “user beware” Funding & time line

14. What gets returned? All remaining tissues & all labels Return an aliquot of extract with specimen? – Extracts must be stored in a (long-term) “tissue bank” – What about costs of storing tissues/extracts? © Natural History Museum

15. What about the DNA? GenBank accession required (within one year?) “All rights reserved” to originating museum Should the researcher also return sequence data? Do museums have databases to manage these data? – Link GenBank accession numbers with catalog numbers – Metadata: extract methods, concentration, location, etc. – DNA sequences for each gene GenBank Nucleotide Search (BLAST)

17. Non-invasive DNA Extraction Techniques for Parasitic Hymenoptera Jeremy C Andersen and Nicholas J Mills University of California Berkeley

18. Questions Can non-invasive techniques be used for parasitic Hymenoptera? Concerns: – small body size – delicate What are effects of size and age of specimen? Can DNA extracts be used for PCR and sequencing?

19. Methods Selection criteria for Braconidae from Essig Museum of Entomology 1) Species differing in adult size 2) Wide range of ages 3) Large number of specimens Atanycolus longifemoralis 15 specimens 1931 – 1981 Meteorus trachynotus 9 specimens 1914 – 2009 * Trioxys pallidus 12 specimens 1959 - 1993

20. DNA Extraction Followed methods and protocols outlined in Gilbert et al. 2007 – Slight modifications to several steps – E-mail: jandersen@berkeley.edu for protocols and/or materials listjandersen@berkeley.edu DNA yield was measured with a NanoDrop spectrophotometer Sequenced 28S and COI genes (long & short) Specimen dried, weighed, and remounted

21. Results A. longifemoralis ~ 4 mg M. trachynotus ~ 0.2 mg T. pallidus ~ 0.01 mg Log(DNA concentraion) 0 1 2 3 4 5 6 7

22. p = 0.019 Age Effect of Specimen age on outcome of PCR (28S) 1 0

23. Discussion Successful extraction of DNA and PCR amplification from specimens back to 1940 Two markers differed in their success rates – Species and Age

24. Comments Discoloration of Body – Reduced percentages of chemical SDS worked for new specimens (Meteorus 2009) with less discoloration Weighing and general handling of insects caused most damage Primer design is extremely important Nanodrop readings (phenol and DNA similar)

25. Useful Literature Pääbo, S. (1989). "Ancient DNA: extraction, characterization, molecular cloning, and enzymatic amplification." Proceedings of the National Academy of Sciences 86(6): 1939-1943. Whitfield, J. B. and S. A. Cameron (1994). "Museum policies concerning specimen loans for molecular systematic research." Molecular Phylogenetics and Evolution 3(3): 268-270. Hafner, M. S. (1994). "Reply: Molecular extracts from museum specimens can—and should—be saved." Molecular Phylogenetics and Evolution 3(3): 270-271. Whitfield, J. B. and S. A. Cameron (1994). "Authors' response to Hafner." Molecular Phylogenetics and Evolution 3(3): 271- 272. Thomas, R. H. (1994). "Molecules, museums and vouchers." Trends in Ecology & Evolution 9(11): 413-414. Whitfield, J. B. (1995). "Museum loan and voucher specimen policies for molecular systematics research." Insect Collection News 10: 3. Phillips, A. J. and C. Simon (1995). "Simple, efficient, and nondestructive DNA extraction protocol for arthropods." Annals of the Entomological Society of America 88(3): 281-283. Golenberg, E. M., A. Bickel, et al. (1996). "Effect of highly fragmented DNA on PCR." Nucleic Acids Research 24(24): 5026-5033. Whitfield, J. B. (1999). Destructive sampling and information management in molecular systematic research: an entomological perspective. Managing the modern herbarium: An interdisciplinary approach. S. Byers and D. Metsger. Ontario, Society for Preservation of Natural History Collections and Royal Ontario Museum: 301-314. Rohland, N., H. Siedel, et al. (2004). "Nondestructive DNA extraction method for mitochondrial DNA analyses of museum specimens." Biotechniques 36(5): 814-821. Gilbert, M. T. P., et al. (2007). "DNA extraction from dry museum beetles without conferring external morphological damage." PLoS ONE 2(3): e272.

26. Acknowlegments Jeremy Anderson (UC Berkeley) Jim Whitfield (Univ. Illinois) David Furth (NMNH/SI) James Cokendolpher (Texas Tech) Katrina Menard (Sam Noble Oklahoma MNH) Chris Marshall (Oregon State University) James Liebherr (Cornell University) Doug Yanega (UC Riverside) Kipling Will (UC Berkeley) Kevin Tuck (Natural History Museum) Dowload this presentation from http://nature.berkeley.edu/~poboyski/http://nature.berkeley.edu/~poboyski/