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Exploring barcode splits among morphologically cryptic species of Lepidoptera through examining alternative loci and next-generation sequencing . Claudia Bertrand, Rodolphe Rougerie , Daniel H. Janzen, Winnie Hallwachs and Mehrdad Hajibabaei. outline. Cryptic species & DNA barcodes Methods

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  1. Exploring barcode splits among morphologically cryptic species of Lepidoptera through examining alternative loci and next-generation sequencing Claudia Bertrand, RodolpheRougerie, Daniel H. Janzen, Winnie Hallwachs and MehrdadHajibabaei

  2. outline • Cryptic species & DNA barcodes • Methods • Case study 1: M. clusoculis, B. perses, E.satellitia • Case study 2: U.belli • Case study 3: E. imperialis

  3. What are cryptic species? “species that are hidden under a single taxonomic name because they are morphologically indistinguishable” (Bickford et al., 2007) • Delineation • Morphology • Ecology • Geography • Behaviour • Genetic janzen.sas.upenn.edu

  4. Astraptesfulgeratorcomplex Hebert, P. D. N., et al, 2004, Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptesfulgerator: PNAS

  5. 52% Morphological or Ecological Correlates 48% Lack Morphological or Ecological Correlates Janzen, D. et al, 2009, Integration of DNA barcoding into an ongoing inventory of complex tropical biodiversity: Molecular Ecology Resources

  6. 1 2 3

  7. outline • Cryptic species & DNA barcodes • Methods • Case study 1: M. clusoculis, B. perses, E.satellitia • Case study 2: U.belli • Case study 3: E. imperialis

  8. Methods: Alternative loci Approach • Mitochondrial markers • Cytochdromec oxidase I (COI, 658bp) • Cytochromeb (500bp) • Nuclear markers • Elongation factor-1-alpha (EF1a, 1000bp), • Internal transcribed spacer II (ITS2, bp vary) • Analysis • Bayesian analysis • Congruence

  9. Methods: 454-Pyrosequencing Method: • MID tagged specimens • Quality filtered & chimera detection • Collapsed into sequence-types • Contig F&R sequence analysis • not possible for Eaclesimperialis or Bardaximaperses Analysis: • NJ-tree (outgroup to divergent) & MP (gaps = fifth character) • Secondary structure & compensatory base change (CBC)

  10. Methods: Wolbachiascreening Wolbachia surface protein (wsp) • Search Wolbachiawspdatabase Multilocus Sequence Typing (MLST) • 5 gene regions used to ID wolbachiastrains • Search wolbachiaMLST database Werren et al ., 2008 Nature Reviews

  11. outline • Cryptic species & DNA barcodes • Methods • Case study 1: M. clusoculis, B. perses, E.satellitia • Case study 2: U.belli • Case study 3: E. imperialis

  12. Case study 1: Mimoidesclusoculis 96 N=10 98 N=10 95 64 77 99 1% 1% Wolbachiaabsent 71 M. clusoculisDHJ01 M. clusoculisDHJ02 69 0.1% 86 74 cytb ITS2 COI EF1a 0.5%

  13. Case study 1: Bardaximaperses 98 N = 32 1 N = 30 99 1 99 0.05% 1% 0.5% 1% Wolbachiaabsent B. persesDHJ01 B. persesDHJ02 cytb ITS2 COI EF1a

  14. Case study 1: Eumorphasatellitia N = 10 • 98 0.2% N = 10 5% • 96 5% • 74 • 84 0.1% Wolbachiaabsent E. satellitiaDHJ01 E. satellitiaDHJ02 E. satellitiaDHJ03 cytb ITS2 COI EF1a

  15. What then, are these mt-lineages? • Recent speciation event • Retention of ancestral polymorphisms in nuclear markers • Find a character that represents species boundaries • 2. Under-detected heteroplasmy or pseudogenes • 454-sequence base approaches for deep sequencing of intra-individual variability • 3. Reticulation instead of speciation • Representative of a past isolation event • Sequence individuals that are allopatrically distributed

  16. outline • Cryptic species & DNA barcodes • Methods • Case study 1: M. clusoculis, B. perses, E.satellitia • Case study 2: U.belli • Case study 3: E. imperialis

  17. Case study 2: Urbanus belli U.belliDHJ02 U.belliDHJ03 Rainforest 100 100 100 1% Dryforest U. belliDHJ01

  18. Case study 2: Urbanus belli W W N=10 100 N=11 W 100 W 100 100 100 W N=12 W W 100 100 100 93 100 100 W 70 Wolbachiapresent 0.5% 0.5% 1% U. belliDHJ02 71 U. belliDHJ03 cytb ITS2 COI EF1a U. belliDHJ01

  19. Wolbachiainfection: Unidirectional Supergroup B Strain: Lepspecies from Ecuador Werren et al ., 2008, Nature Reviews

  20. ITS2 secondary structure: Compensatory base changes (CBC) I IV II (marked by U-U mismatch) All compensatory base changes were found in helices II and III III Interspecific CBC >> Intraspecific CBC 51% consensus of aligned structures with gaps

  21. outline • Cryptic species & DNA barcodes • Methods • Case study 1: M. clusoculis, B. perses, E.satellitia • Case study 2: U.belli • Case study 3: E. imperialis

  22. Case study 3: Eaclesimperialis Rainforest 100 100 DHJ02 DHJ01 Semi-deciduous lowland forest 1% Dryforest

  23. 46 14 4 29 8 8 2 30 2 0 5 0 1 1 1 5 1 0 0 0 0 0 1 15 0 0 0 0 0 3 0 0 0 0 4 0 7 0 21 0 0 0 0 0 0 0 0 0 44 24 0 0 0 33 0 0 0 32 0 63 0 0 0 0 61 0 0 0 0 0 0 0 15 0 46 0 0 0 0 0 0 0 50 0 0 0 0 64 45 1 16 7 38 1 3 0 3 31 0 1 0 0 47 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 50 0 12 0 0 0 1 0 0 0 0 50 0 0 42 1 21 3 7 51 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 63 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 17 28 0 0 34 45 0 0 4 0 60 23 0 0 0 62 0 0 0 0 0 1 0 0 1 0 6 10 0 0 0 53 0 0 0 0 41 16 19 0 26 18 0 5 0 0 25 0 1 0 1 0 2 0 0 0 0 0 1 2 5 3 2 1 0 0 3 0 0 0 0 3 0 0 0 3 0 0 0 1 0 0 0 0 0 43 0 44 0 6 1 36 0 0 0 0 0 0 0 0 0 0 0 8 0 0 0 0 0 0 0 0 0 0 0 0 9 0 7 7 13 0 13 13 0 0 0 0 10 0 0 10 29 0 0 0 1 2 9 10 1 6 5 6 8 61 9 15 4 7 8 9 23 10 8 29 63 26 26 26 1 3 1 18 0 23 31 0 0 0 0 0 0 38 43 0 27 0 0 0 63 0 0 0 0 0 0 0 2 41 1 3 4 4 1 6 5 29 8 9 2 41 0 10 0 7 7 9 10 24 29 Case study 3: Eaclesimperialis 0.2% Retention of ancestral polymorphisms 2. Interbreeding 2% 2% 1% Wolbachiaabsent E. imperialisDHJ01 E. imperialisDHJ02 cytb ITS2 COI EF1a

  24. Case study 3 COI 1% DHJ02 1. non-sister status 2. ACG secondary contact zone South American North American DHJ01

  25. Case study 3 EF1a 1% Eacles imperialisDHJ01/DHJ02

  26. Case Study 3 ITS2 (sanger) 165 167 170 172-177 179 117-127 80 140 155 DHJ01/ DHJ02 N. America S. America

  27. Conclusions to date: • ACGsecondary contact zone • Interbreeding • Parapatric– female ecological preferences • Male-driven – COI lineages

  28. Acknowledgement Collaborators Committee Dr. Dan Janzen Dr. Winnie Hallwachs Dr. RodolpheRougerie Dr. Alex Smith Dr. Teresa Crease Hajibabaei Lab Funding ShadiShokralla Joel Gibson Ian King SainaTaidi Claudia Bertrand Jennifer Spall Stephanie Boilard Steven Van Konynenburg Melissa Braschel Jessica Klawunn Vanessa Patterson-Doherty Mehrdad Hajibabaei Genome Canada Ontario Genomics Institute NSERC Canada

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