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

2

outline

1. Cryptic species & DNA barcodes2. Methods3. Case study 1: M. clusoculis, B. perses, E.satellitia4. Case study 2: U.belli5. 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

Astraptes fulgerator complexHebert, P. D. N., et al, 2004, Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator: PNAS

5Janzen, D. et al, 2009, Integration of DNA barcoding into an ongoing inventory of complex tropical biodiversity: Molecular Ecology Resources

52% Morphological or Ecological Correlates

48% Lack Morphological or Ecological Correlates

Family Species Larval

foodplant Ecosystem Adult

morphology % Barcode Divergence

Distribution(ACG)

Papilionidae Mimoides clusoculisDHJ01

Overlapping Dryforest, Rainforest

Overlapping 1% Sympatric

Mimoides clusoculisDHJ02

Dryforest, Rainforest

Notodontidae Bardaxima persesDHJ01 Overlapping Dryforest, Rainforest

Overlapping 2% Sympatric

Bardaxima persesDHJ02 Dryforest, Rainforest

Sphingidae Eumorpha satellitiaDHJ01

Overlapping Dryforest, Rainforest

Overlapping 2-4% Sympatric

Eumorpha satellitiaDHJ02

Dryforest, Rainforest

Eumorpha satellitiaDHJ03

Dryforest, Rainforest

Hesperiidae Urbanus belliDHJ01 Overlapping Dryforest, Rainforest

Overlapping 3-4% Sympatic

Urbanus belliDHJ02 Dryforest, Rainforest

Urbanus belliDHJ03 Rainforest

Saturniidae Eacles imperialisDHJ01 Overlapping Rainforest Larger female genitalia

8% Parapatric

Eacles imperialisDHJ02 Dryforest

6

1

2

3

7

outline

1. Cryptic species & DNA barcodes2. Methods3. Case study 1: M. clusoculis, B. perses, E.satellitia4. Case study 2: U.belli5. Case study 3: E. imperialis

8

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

Methods:

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454-PyrosequencingMethod:• 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)

Methods:

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Wolbachia screening

Wolbachia surface protein (wsp) • Search Wolbachia wsp database

Multilocus Sequence Typing (MLST)• 5 gene regions used to ID wolbachia strains• Search wolbachia MLST database

Methods:

Werren et al ., 2008Nature Reviews

11

outline

1. Cryptic species & DNA barcodes2. Methods3. Case study 1: M. clusoculis, B. perses, E.satellitia4. Case study 2: U.belli5. Case study 3: E. imperialis

12

Case study 1: Mimoides clusoculis

0.1%74

8669

71

64

0.5%

N=10

N=10

99

95

96

1%

77

98

1%

COI cytb ITS2EF1a

M. clusoculisDHJ01M. clusoculisDHJ02

Wolbachia absent

13

1% 0.5%

N = 32

N = 30

99

99

98

1%

1

0.05%

1

Case study 1: Bardaxima perses

COI cytb ITS2EF1a

B. persesDHJ01B. persesDHJ02

Wolbachia absent

14

Case study 1: Eumorpha satellitia

E. satellitiaDHJ01E. satellitiaDHJ02E. satellitiaDHJ03

COI cytb ITS2EF1a

5%

N = 10

N = 10

5%

0.1%

0.2%

98

84

96

74

Wolbachia absent

15

What then, are these mt-lineages?

3. Reticulation instead of speciation• Representative of a past isolation event• Sequence individuals that are allopatrically distributed

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

16

outline

1. Cryptic species & DNA barcodes2. Methods3. Case study 1: M. clusoculis, B. perses, E.satellitia4. Case study 2: U.belli5. Case study 3: E. imperialis

17

Case study 2: Urbanus belli U.belliDHJ03 U.belliDHJ02 U. belliDHJ01

1%

100

100

100

Dryforest

Rainforest

18

Case study 2: Urbanus belli

U. belliDHJ02 U. belliDHJ03

U. belliDHJ01

COI cytb ITS2EF1a

0.5%

100

93

71

70

0.5%

W

WW

W

W

W

W

W

100

100

100 100

100

100

N=10

N=11

N=12

1%

100

100

100

Wolbachia present

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Marker Allele% Identity Match in WSP &

MLST Databasewsp 115 100%gatB 71 98%coxA 67 100%ftsZ 65 99%

hcpA 74 99%fbpA 6 98%

Supergroup B Strain:

Lep species from Ecuador

Wolbachia infection: Unidirectional

Werren et al ., 2008, Nature Reviews

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ITS2 secondary structure: Compensatory base changes (CBC)

I

II (marked by U-U mismatch)

III

IV

51% consensus of aligned structures with gaps

All compensatory base changes were found in helices II and III

Interspecific CBC >> Intraspecific CBC

21

outline

1. Cryptic species & DNA barcodes2. Methods3. Case study 1: M. clusoculis, B. perses, E.satellitia4. Case study 2: U.belli5. Case study 3: E. imperialis

22

Case study 3: Eacles imperialis

Semi-deciduous lowland forest

100

100

1%

DHJ01

DHJ02

Dryforest

Rainforest

23

Case study 3: Eacles imperialis

2%

1%

2%

2 2 0 0 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 00 00000000000000000000000000000000000000000

1448521

3244

1546

16713

73100010

00000

0013000

1

214251

050

50

45236228346017000000

138

1333221021010000

0000000000000000000000000000000000000000000000

0

0

0

43

61

43

5

3129

137

6

03

382763

6329108

7

4118

26

1329

10

2

29

24

10

97 7

00 9 8

5 6 1 4 4 3 120 00000000

000000000131

269

00

0

0

0

1

04

12

3262315101 987498610

5610

9210000000000000000000000000000000

000000000000

000000015

0

3644

261619182541 53 0000010611

000

20

41000

50

1220

81551134724336145

3831470000000000000000000000000

00000000000000000000000000000

0

0 0 0

0

0

64

2

63

30

46

29

63

0.2%

E. imperialisDHJ01 E. imperialisDHJ02

COI cytb ITS2EF1a

Wolbachia absent

1. Retention of ancestral polymorphisms

2. Interbreeding

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Case study 3COI

DHJ01

DHJ02

North American

South American

1%

1. non-sister status

2. ACG secondary contact zone

251%

Eacles imperialisDHJ01/DHJ02

Case study 3EF1a

26

09-SRNP-1430909-SRNP-1431009-SRNP-6557609-SRNP-142382006-ONT-09662006-ONT-096506-FLOR-023806-FLOR-030506-FLOR-051106-FLOR-030406-FLOR-1393BC-Her1855BC-Her1235BC-Her1853BC-Her1253BC-Dec1104BC-Her1366BC-Her1364BC-Her1370BC-EvS 1936BC-FMP-1428BC-RBP 4611BC-Dec0120BC-Dec0446BC-Her0458BC-Dec0448BC-Dec0445BC-RBP 4604BC-Her1704BC-Dec0146BC-Dec0333

N. America

S. America

80 117-127 140 155 165 167 170 172-177 179

Case Study 3ITS2 (sanger)

DHJ01/DHJ02

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Conclusions to date:

• ACG secondary contact zone

• Interbreeding

• Parapatric – female ecological preferences

• Male-driven – COI lineages

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Acknowledgement Collaborators CommitteeDr. Dan JanzenDr. Winnie HallwachsDr. Rodolphe RougerieDr. Alex SmithDr. Teresa Crease

Hajibabaei LabShadi Shokralla Joel Gibson Ian King Saina Taidi Claudia BertrandJennifer Spall Stephanie BoilardSteven Van Konynenburg Melissa Braschel Jessica Klawunn Vanessa Patterson-DohertyMehrdad Hajibabaei

FundingGenome CanadaOntario Genomics InstituteNSERC Canada