1. Success of DNA barcoding in distinguishing sister species of diverse clades of birds Allan Baker, Erika Tavares, Rebecca Elbourne Department of Natural History, Royal Ontario Museum

2. Organization of the talk (1) Sister-species comparisons need to be tested (2)10X rule for the ratio of intra- to interspecific distance fails to detect sister and cryptic species (3) Multiple genes are needed to barcode species uniquely (4) More extensive sampling of intra-specific diversity (5) Problems with hybridization and paralogy in birds Aim is to address the following concerns about DNA barcoding:

3. Barcoding sister-species of birds Multigene phylogenies Sister-species selection COI alignment DNA extraction Amplification and sequencing of COI GenBank and BOLD search Calculate COI corrected distances Map COI of sister- species in multigene phylogeny

4. Multigene phylogeny of terns * Posterior Prob. = 1.00 Bayesian analysis 12S, Cytb, ND2, COI :3320 bp Bridge et al 2005, MPE 35: 459-469 COI:651 bp Distances

5. Multigene phylogeny of shanks Bayesian analysis 12S and 16S rDNAs, ATPase8, ATPase6, COIII, ND1, tRNA-ISO, ND2, ND5, Cytb: 7664 bp. Pereira and Baker 2005 Condor 107. COI:651 bp Distances * Posterior Prob. = 1.00

6. Multigene phylogeny of Calidridine sandpipers COI:~763 bp Distances Bayesian analysis RAG1 + mtDNA: 12,143 bp. Baker et al. In prep.

7. Multigene phylogeny of penguins COI:~615 bp Distances Bayesian analysis RAG1, 12S, 16S, COI, Cytb: 5571 bp. Pereira and Baker et al (2006) Proc. Biol. Sci 273. * Posterior Prob. = 1.00

8. Multigene phylogeny of kiwis COI:~650 bp Distances Maximum Likelihood ATPAse 6, ATPase 8, CR, Cytb: 2267 bp. Burbidge et al (2003) Conservation Genetics 4. * Bootstrap proportion = 100%

9. Multigene phylogeny of Pteroglossus toucans COI:~615 bp Distances Bayesian analysis ATPase, COI, Cytb: 2168 bp. Eberhard and Bermingham (2005) MPE: 288-304 * Posterior Prob. = 1.00

10. Multigene phylogeny of Woodpeckers Bayesian analysis COI, Cytb: 2580 bp. Moore et al (2006) Biol. J. Linn. Soc.87 * Posterior Prob. = 1.00 COI:~660 bp Distances

11. Sister-species barcode comparisons Do many genes improve on COI barcoding of sister-species in 7 clades of birds? Multigene phylogenies Sister-species selection COI alignment of well sampled species DNA extraction Amplification and sequencing of COI GenBank and BOLD search Calculate COI distances and NJ topologies: K2P Test barcode in COI vs multigenes

12. Success in barcoding sister-species of birds Sample of 812 sequences from 95 sister-species pairs 2 Clusters1 Cluster COI93.7% (89)6.3%(6) Multigene93.7% (89)6.3%(6) Of the 6 “failures” 4 were known to be from hybridization Conclusion: No improvement using more genes

13. Intraspecific versus among sister-species variation Sample of 812 sequences from 95 sister-species pairs. Conclusion: This is a potential problem for barcoding sister-species?

14. Within-species variation in terns Terns Intra-specific Between sister-species Within genera (above sister species level) Between genera Neighbour Joining (Kimura-2P) Node support: Posterior Prob. Bayesian analysis. COI (651 bp) Multigene 3220 bp)

15. New species of Little terns? 1.5% 1.3%

16. http://www.birdguides.com Large-bill form Small-bill form 1.7% 1.8% New species of Gull-billed terns? Gull-billed Tern

17. Within-species variation in Tringid sandpipers Intra-specific Between sister-species Within genera (above sister species level) Between genera Neighbour Joining (model Kimura-2P) Node support: Posterior Prob. Bayesian analysis. COI (623 bp) Multigene (7664 bp) *

18. Intra-specific Between sister-species Within genera (above sister species level) Between genera Within-species variation in penguins Penguins COI (bp) Multigene (bp) Neighbour Joining (model Kimura-2P) Node support: Posterior Prob. Bayesian analysis.

19. 2- White-Flippered Penguin Eudyptula m. albosignata (J.N. Davis and J.Busby) 3-Eudyptula m. novaehollandiae (J.N. Davis and J.Busby) 1-Little Penguin Eudyptula m. minor (J.N. Davis and J.Busby) a,b c,d a,b e,f g,h Banks, JC. 2002 Notornis 49:29-38. Northern a Northern b Banks Peninsula b Banks Peninsula a Cook Strait c Cook Strait d Chatham Is. e Chatham Is. f Otago g Otago h Australia a Australia b COI barcode 3.56% Total Evidence Tree New species of Little blue penguins 1%

20. New species of kiwis COI 650 bp Multigene phylogeny

21. Threshold for sister-species Excluding putative new species and hybridizing species

22. Threshold for sister-species

23. Conclusion: Sister-species and intraspecific variation overlap slightly around 1% level Threshold for sister-species

24. COI barcodes vs time of divergence of species Estimation of divergence times if not available with multidivtime Multigene phylogenies COI alignment COI distances vs divergence times Model choice

25. Species barcode level varies with time since speciation

26. Summary for these clades The levels of distances between sister-species pairs varies from 1% to more than 20%. When putative new species and hybridizing forms are excluded, COI distances (>1.3% ) between sister-species do not overlap with intraspecific variation However, all sister-species have unique DNA barcodes that identify them Results are specific to the clades we investigated, and potentially will vary with different rates of evolution in birds with faster rates of evolution

28. These phenomena can be flagged with COI barcoding By exposing these difficult cases barcoding will speed higher resolution studies required to make taxonomic decisions on species limits Most species lose the ability to hybridize within 1-2 million years after divergence, so it should not be a pervasive problem in the tree of life Even in closely related sister-species of birds less than 6% hybridize, so barcoding is highly effective in cataloguing species diversity and discovering putative new species. Hybridization, unsorted polymorphism and paralogy

29. Acknowledgements This research was supported by funding to the Canadian Barcode of Life Network from Genome Canada through the Ontario Genomics Institute, NSERC, and other sponsors listed at www.BOLNET.ca.www.BOLNET.ca We thank Oliver Haddrath, Sergio Pereira, Eli Bridge and the Hebert lab for tips and assistance, and Rob Brumfield,Joel Cracraft and Bob Zink for permission to barcode loaned samples.