Exploring cryptic Lepidoptera species through DNA barcoding and next-generation sequencing
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This document summarizes research examining cryptic species of Lepidoptera using DNA barcoding and next-generation sequencing. Three case studies are presented: 1) Three cryptic species were found within Mimoides clusoculis and Eumorpha satellitia using alternative loci and Wolbachia screening. 2) Urbanus belli was split into three cryptic species with Wolbachia infections correlating with geography. 3) Eacles imperialis showed two cryptic lineages with a contact zone and evidence of interbreeding from nuclear markers. The research demonstrates the utility of DNA barcoding and next-generation sequencing to detect cryptic diversity within morphologically similar species.
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Exploring cryptic Lepidoptera species through DNA barcoding and next-generation sequencing
- 1. 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 1
- 2. outline 1. Cryptic species & DNA barcodes 2. Methods 3. Case study 1: M. clusoculis, B. perses, E.satellitia 4. Case study 2: U.belli 5. Case study 3: E. imperialis 2
- 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 3 janzen.sas.upenn.edu
- 4. Astraptes fulgerator complex 4 Hebert, P. D. N., et al, 2004, Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator: PNAS
- 5. 52% Morphological or Ecological Correlates 48% Lack Morphological or Ecological Correlates 5 Janzen, D. et al, 2009, Integration of DNA barcoding into an ongoing inventory of complex tropical biodiversity: Molecular Ecology Resources
- 6. Larval Adult % Barcode Distribution Family Species foodplant Ecosystem morphology Divergence (ACG) Papilionidae Mimoides Overlapping Dryforest, Overlapping 1% Sympatric clusoculisDHJ01 Rainforest Mimoides Dryforest, clusoculisDHJ02 Rainforest Notodontidae Bardaxima persesDHJ01 Overlapping Dryforest, Overlapping 2% Sympatric Rainforest 1 Bardaxima persesDHJ02 Dryforest, Rainforest Sphingidae Eumorpha Overlapping Dryforest, Overlapping 2-4% Sympatric satellitiaDHJ01 Rainforest Eumorpha Dryforest, satellitiaDHJ02 Rainforest Eumorpha Dryforest, satellitiaDHJ03 Rainforest Hesperiidae Urbanus belliDHJ01 Overlapping Dryforest, Overlapping 3-4% Sympatic Rainforest Urbanus belliDHJ02 Dryforest, 2 Rainforest Urbanus belliDHJ03 Rainforest Saturniidae Eacles imperialisDHJ01 Overlapping Rainforest Larger female 8% Parapatric 3 genitalia Eacles imperialisDHJ02 Dryforest 6
- 7. outline 1. Cryptic species & DNA barcodes 2. Methods 3. Case study 1: M. clusoculis, B. perses, E.satellitia 4. Case study 2: U.belli 5. Case study 3: E. imperialis 7
- 8. Methods: Alternative loci Approach Mitochondrial markers • Cytochdrome c oxidase I (COI, 658bp) • Cytochrome b (500bp) Nuclear markers • Elongation factor-1-alpha (EF1a, 1000bp), • Internal transcribed spacer II (ITS2, bp vary) Analysis • Bayesian analysis • Congruence 8
- 9. Methods: 454-Pyrosequencing Method: • MID tagged specimens • Quality filtered & chimera detection • Collapsed into sequence-types • Contig F&R sequence analysis – not possible for Eacles imperialis or Bardaxima perses Analysis: • NJ-tree (outgroup to divergent) & MP (gaps = fifth character) • Secondary structure & compensatory base change (CBC) 9
- 10. Methods: Wolbachia screening Wolbachia surface protein (wsp) • Search Wolbachia wsp database Werren et al ., 2008 Multilocus Sequence Typing (MLST) Nature Reviews • 5 gene regions used to ID wolbachia strains • Search wolbachia MLST database 10
- 11. outline 1. Cryptic species & DNA barcodes 2. Methods 3. Case study 1: M. clusoculis, B. perses, E.satellitia 4. Case study 2: U.belli 5. Case study 3: E. imperialis 11
- 12. Case study 1: Mimoides clusoculis 96 N=10 98 N=10 95 64 77 99 1% M. clusoculisDHJ01 1% M. clusoculisDHJ02 71 69 Wolbachia absent 0.1% 86 0.5% 74 COI cytb EF1a ITS2 12
- 13. Case study 1: Bardaxima perses 98 N = 32 1 N = 30 99 1 99 0.05% 1% 1% 0.5% B. persesDHJ01 B. persesDHJ02 Wolbachia absent COI cytb EF1a ITS2 13
- 14. Case study 1: Eumorpha satellitia 98 N = 10 0.2% 96 74 84 N = 10 5% 5% E. satellitiaDHJ01 0.1% E. satellitiaDHJ02 Wolbachia absent E. satellitiaDHJ03 COI cytb EF1a ITS2 14
- 15. What then, are these mt-lineages? 1. 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 15
- 16. outline 1. Cryptic species & DNA barcodes 2. Methods 3. Case study 1: M. clusoculis, B. perses, E.satellitia 4. Case study 2: U.belli 5. Case study 3: E. imperialis 16
- 17. Case study 2: Urbanus belli U.belliDHJ03 U.belliDHJ02 U. belliDHJ01 100 Rainforest 100 100 1% Dryforest 17
- 18. Case study 2: Urbanus belli 100 W 100 N=10 W 100 N=11 100 W 100 W N=12 W 93 100 100 W 100 W 100 100 70 W 1% 0.5% 0.5% 71 U. belliDHJ03 U. belliDHJ02 Wolbachia present U. belliDHJ01 COI cytb EF1a ITS2 18
- 19. Wolbachia infection: Unidirectional % Identity Match in WSP & Marker Allele MLST Database wsp 115 100% gatB 71 98% coxA 67 100% ftsZ 65 99% hcpA 74 99% fbpA 6 98% Supergroup B Strain: Werren et al ., 2008, Nature Reviews Lep species from Ecuador 19
- 20. ITS2 secondary structure: Compensatory base changes (CBC) I IV II (marked by U-U mismatch) All compensatory base changes III were found in helices II and III Interspecific CBC >> Intraspecific CBC 51% consensus of aligned structures with gaps 20
- 21. outline 1. Cryptic species & DNA barcodes 2. Methods 3. Case study 1: M. clusoculis, B. perses, E.satellitia 4. Case study 2: U.belli 5. Case study 3: E. imperialis 21
- 22. Case study 3: Eacles imperialis 100 DHJ02 Rainforest DHJ01 100 Semi-deciduous lowland forest Dryforest 1% 22
- 23. Case study 3: Eacles imperialis 2 1. Retention of ancestral polymorphisms 1% 0 0 2% 2. Interbreeding 0 2% 0.2% Wolbachia absent E. imperialisDHJ01 E. imperialisDHJ02 COI cytb EF1a ITS2 23
- 24. Case study 3 COI DHJ02 1. non-sister status South American 2. ACG secondary contact zone North American DHJ01 1% 24
- 25. Case study 3 EF1a Eacles imperialisDHJ01/DHJ02 1% 25
- 26. Case Study 3 ITS2 (sanger) 80 117-127 140 155 165 167 170 172-177 179 DHJ01/ 09-SRNP-14309 09-SRNP-14310 09-SRNP-65576 DHJ02 09-SRNP-14238 2006-ONT-0966 2006-ONT-0965 06-FLOR-0238 06-FLOR-0305 06-FLOR-0511 06-FLOR-0304 06-FLOR-1393 N. America BC-Her1855 BC-Her1235 BC-Her1853 BC-Her1253 BC-Dec1104 BC-Her1366 BC-Her1364 BC-Her1370 BC-EvS 1936 BC-FMP-1428 BC-RBP 4611 BC-Dec0120 BC-Dec0446 BC-Her0458 S. America BC-Dec0448 BC-Dec0445 BC-RBP 4604 BC-Her1704 BC-Dec0146 BC-Dec0333 26
- 27. Conclusions to date: • ACG secondary contact zone • Interbreeding • Parapatric – female ecological preferences • Male-driven – COI lineages 27
- 28. Acknowledgement Hajibabaei Lab Collaborators Funding Shadi Shokralla Committee Genome Canada Joel Gibson Dr. Dan Janzen Ontario Genomics Institute Ian King Dr. Winnie Hallwachs NSERC Canada Saina Taidi Dr. Rodolphe Rougerie Claudia Bertrand Dr. Alex Smith Jennifer Spall Dr. Teresa Crease Stephanie Boilard Steven Van Konynenburg Melissa Braschel Jessica Klawunn Vanessa Patterson-Doherty Mehrdad Hajibabaei 28
Editor's Notes
- I)M)S) Add caterpillarsMsc -- Hajibabaei lab at UofGPrivileged to share my master’s thesis About exploring barcode splits of morphologically cryptic species of Lepidoptera
- I)What are cryptic species?M)really a bi-product of how we detect species which has historically been by morphological identification S) as we integrate different datasets like natural history data (specifically for leps, caterpillar morphology, host plant specificity, genitalic morphology, molecular data what we thought was a single evolutionary lineage can change.
- I)Specific examples of cryptic species that were initially underscored by DNA barcodes are:M) Ten species in one, Perichares, S) . Key discoveries in the Hesperiidae family include the once polyphagousAstraptesfulgerator, now divided into 10 cryptic species based on DNA barcode divergences, distinct food-plants, subtle morphological traits and ecological preferences(Hebert et al., 2004).Pericharesphiletesis now divided into four species described by DNA barcode divergences as well as caterpillars, pupae and food-plant differences (Burns et al., 2008).Most recently, Burns et al. discovered crypsis within Porphyrogenespeterwegei, using DNA barcode divergences, caterpillar developmental changes, food-plant conservatism and host-specific parasitism (2010).
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- To further investigate this case we expanded our sampling to include ind from geographically distributed populationsTo try and assertain if DHJ01/DHJ02 are sister taxa, which would suggest this incongruence to be incomplete lineage sorting of nuclear alleles. However if they turn out not to be sister taxa then the ACG is a secondary contact zone for these populations and would suggest interbreeding.
- The COI data suggests a
- -ITS2,sanger reads not 454, poor quality due to non-homogenized repeats.-character analysis shows that DHJ01 and DHJ02 share nucleotide characters that are most similar to N American grouping and different from S american grouping.
- Basically ACG represents the place where the two, long-separated lineages meet... there they do not seem to have full reproductive isolation as revealed by introgression of nuclear markers, but they do remain parapatric because of ecological preferences as you suggested - and females being very sedentary, then most if not all of the introgression is male-driven - which explains that nuclear alleles filter through the barrier whereas there's no mitochondrial exchanges...