DNA barcoding of the Lepidoptera of Area de Conservacion Guanacaste, Costa Rica

DNA barcoding of Lepidoptera of the Area de Conservacion

Guanacaste (ACG), Costa Rica

•John J. Wilson

PhD Advisor: Paul Hebert

Collaborators: Mehrdad Hajibabaei, Dan Janzen, Winnie Hallwachs

BIOLEP

Why Barcode Tropical

Lepidoptera???

Current state of Lepidoptera taxonomy

Worldwide distributed, charismatic group with extensive previous taxonomic attention - yet still unknown

165,000 described species out of estimated 280,000 - 1.4 million global total

Higher taxonomic relationships are still “shrouded in mystery”

Macrolepidoptera represent a benchmark group for the ‘encyclopaedia of life’

Area de Conservacion Guanacaste

9,600 species of Macrolepidoptera

As many species as in North America

156,000 hectare reserve

Area de Conservacion Guanacaste

DNA barcodes facilitate:

-Species identifications

-Species discoveries

-Exploration of higher taxonomic relationships

Mission of BioLep:Complete inventory of non-leaf-mining lepidopteran species in the ACG

DNA barcoding

Morphology

DNA Barcode

AACATTATATTTTATTTTTGGAATTTGAGCAGGTATAATTGGAACTTCTC

TAAGTTTATTAATTCGAGCAGAATTAGGTAACCCCGGATCACTAATTGGG

GATGATCAAATTTATAATACTATTGTAACAGCTCATGCATTTATTATAAT

TTTTTTTATAGTTATACCAATTATAATTGGAGGATTTGGAAATTGATTAA

TTCCCCTTATATTAGGGGCCCCTGACATAGCTTTTCCGCGAATAAATAAT

ATAAGATTTTGATTATTACCCCCTTCTATTATATTATTAATTTCAAGAAG

AATTGTAGAAAATGGAGCAGGAACTGGATGAACTGTGTACCCACCTTTAT

CCTCAAATATTGCACATGGGGGAAGATCTGTAGATTTAGCAATTTTTTCC

TTACATTTGGCGGGAATTTCATCAATTTTAGGGGCTATTAATTTTATTAC

AACAATTATTAATATACGATTAAATAACATATCATTTGATCAAATACCAT

TATTTGTTTGAGCTGTGGGAATTACTGCATTTTTACTTTTACTTTCATTG

CCTGTTTTAGCGGGAGCTATTACTATATTATTAACAGATCGAAATTTAAA

TACTTCATTTTTTGATCCTGCTGGAGGAGGTGACCCTATTTTATATCAAC

ATTTATTT

DNA Barcode

Barcode collection: 1

100 km

heavy caterpillar inventoryheavy light trapping


www.boldsystems

.org

Area de Conservación Guanacaste

Light trapping of specimens

DNA Barcode

ACTGTCTA…

DNA extraction

PCR

Sequencing

Photo

Specimen

Legsample

University of Guelph

GenBank

GPS record

Applying DNA barcoding to the taxonomic inventory

Species identification

Species identification: popular approach

Phenetic Thresholds“Genetic Yardstick”

2%

Species Identification: test family Saturniidae

170 Saturniid BioLep Specimens (37 species)

BOLDwww.boldsystems.orgClosest match in ‘Reference’ dataset within a limit of 5% sequence divergence

GenBankwww.ncbi.nlm.nih.govBest BLAST ‘hit’

Saturniidae has good coverage in public databases due to published project of Hajibabaei et al. (2006) PNAS - 73 ACG species.

57.6

62.4

5.3

37.637.1

0.00.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

BOLD GenBank

Database Search

Pe

rce

nta

ge

Correct ID

False Positive

Unknown

Species identification: alternative approaches

Characteristic Attribute Organization System

1. Cladogram

0

1

10

0 1

NODE GROUP POS STATE CONF0 0 90 C 10 0 171 T 10 1 90 T 10 1 171 A 11 0 108 A 11 0 153 T 11 1 108 T 11 1 153 A 1

Nucleotide Position Taxa 90 108 153 171

A C T A TB T T T AC T A A A

2. Search for characteristic attributes with CAOS algorithm

3. Find unique combinations of character states

Species identification: alternative approaches

MaximumLikelihood



Species discovery

Species discovery: hypothesis generation

Species discovery: hypothesis testing

Represents a generalization from which future observations are predicted.

When future observations are made, the hypothesis is either corroborated or refuted.

Species discovery hypotheses are subject to testing through concordance with independent sources of data.

Every putative species is a research hypothesis

Species discovery: hypothesis testing

Baum & Shaw (1995)

Species discovery: test family Nymphalidae

15 putative species identified by the barcode sequence pattern

(from the ACG reared project)

Next steps:

•Sequencing nuclear gene regions -wingless, ITS+28S, other nuclear introns

•Morphology – genitalia dissections

Future steps:

•Include specimens from wider geographical area

•Compare with type specimens

Adelpha basiloidesDHJ01Adelpha basiloidesDHJ01Adelpha basiloidesDHJ01

Adelpha basiloidesDHJ01Adelpha basiloidesDHJ01Adelpha basiloidesDHJ02Adelpha basiloidesDHJ02Adelpha basiloidesDHJ02Adelpha basiloidesDHJ02Adelpha basiloidesDHJ02Adelpha cytherea

Adelpha melantheAdelpha phylaca

Adelpha zinaDHJ01Adelpha zinaDHJ01Adelpha zinaDHJ01Adelpha zinaDHJ01

Adelpha zinaDHJ01Adelpha zinaDHJ02Adelpha zinaDHJ02

Adelpha zinaDHJ02Taygetis andromedaDHJ01Taygetis andromedaDHJ01Taygetis andromedaDHJ01Taygetis andromedaDHJ01

Taygetis andromedaDHJ02Taygetis andromedaDHJ02Taygetis andromedaDHJ02Taygetis andromedaDHJ02Taygetis andromedaDHJ02

Taygetis virgiliaTaygetis kerea

Memphis aulicaMemphis cleomestra

Memphis forreriDHJ03Memphis forreriDHJ03Memphis forreriDHJ03Memphis forreriDHJ03Memphis forreriDHJ03

Memphis forreriDHJ04Memphis forreriDHJ04Memphis forreriDHJ04

Memphis forreriDHJ04Memphis forreriDHJ04Memphis moruusDHJ01Memphis moruusDHJ01Memphis moruusDHJ01Memphis moruusDHJ01Memphis moruusDHJ01Memphis moruusDHJ02Memphis moruusDHJ02Memphis moruusDHJ02Memphis moruusDHJ02Memphis moruusDHJ02

Zaretis isidoraDHJ01Zaretis isidoraDHJ01Zaretis isidoraDHJ01Zaretis isidoraDHJ01Zaretis isidoraDHJ01


Siderone marthesiaSiderone marthesiaSiderone marthesiaSiderone marthesiaSiderone marthesiaSiderone marthesiaSiderone marthesiaSiderone marthesiaSiderone marthesiaSiderone marthesiaSiderone marthesiaSiderone marthesiaPrepona laertesDHJ01Prepona laertesDHJ01

Prepona laertesDHJ02Prepona laertesDHJ02

Biblis hyperiaDHJ01Biblis hyperiaDHJ01Biblis hyperiaDHJ01Biblis hyperiaDHJ01Biblis hyperiaDHJ01

Biblis hyperiaDHJ02Biblis hyperiaDHJ02Biblis hyperiaDHJ02


Chlosyne Janzen01Chlosyne Janzen01Chlosyne Janzen01Chlosyne Janzen01

Chlosyne Janzen01Chlosyne Janzen02Chlosyne Janzen02Chlosyne Janzen02Chlosyne Janzen02Chlosyne laciniaDHJ01Chlosyne laciniaDHJ01Chlosyne laciniaDHJ02

Chlosyne laciniaDHJ02Chlosyne laciniaDHJ02Chlosyne laciniaDHJ02Chlosyne laciniaDHJ02

Chlosyne gaudialisChlosyne erodyle

Pteronymia latillaDHJ01Pteronymia latillaDHJ01Pteronymia latillaDHJ01

Pteronymia latillaDHJ02Pteronymia latillaDHJ02Pteronymia latillaDHJ02Pteronymia latillaDHJ02Pteronymia latillaDHJ02

Pteronymia pictaDynamine mylittaDHJ01Dynamine mylittaDHJ01

Dynamine mylittaDHJ02Dynamine mylittaDHJ02Dynamine mylittaDHJ02Dynamine mylittaDHJ02

Dynamine mylittaDHJ02Dynamine sosthenes

Caligo atreusCaligo eurilochusCaligo telamoniusDHJ01

Caligo telamoniusDHJ01Caligo telamoniusDHJ01

Caligo telamoniusDHJ01Caligo telamoniusDHJ01Caligo telamoniusDHJ02Caligo telamoniusDHJ02Caligo telamoniusDHJ02Caligo telamoniusDHJ02Caligo telamoniusDHJ02

5 changes

Caligotelamonius(Felder & Felder 1862)

Dynamine mylitta(Cramer 1782)

Pteronymialatilla ( Hewitson[1855])

Chlosyne lacinia(Geyer 1837)

ChlosyneJanzen 1 & 2

Biblis hyperia(Cramer 1782)

Prepona laertes(Hubner [1811])

Memphis forreri(Godman & Salvin 1884)

Sideronemartesia(Cramer 1777)

Zaretis isidora(Cramer 1779)

Memphis moruus( Fabricius 1775)

Taygetisandromeda(Cramer 1779)

Adelpha zina( Hewitson 1867)

Adelphabasiloides(Bates 1865)

Figure 3: One of 200 most parsimonius cladograms inferred from barcode sequences showing 15 potential new Nymphalidae species. Converted to a phylogram to visualize number of nucleotide changes supporting the branch es.














Zaretis isidoraDHJ02















Siderone marthesiaSiderone marthesiaSiderone marthesiaSiderone marthesiaSiderone marthesiaSiderone marthesiaSiderone marthesiaSiderone marthesiaSiderone marthesiaSiderone marthesiaSiderone marthesiaSiderone marthesiaPrepona laertesDHJ01Prepona laertesDHJ01

Prepona laertesDHJ02Prepona laertesDHJ02


Biblis hyperiaDHJ02Biblis hyperiaDHJ02Biblis hyperiaDHJ02


Chlosyne Janzen01Chlosyne Janzen01Chlosyne Janzen01Chlosyne Janzen01

Chlosyne Janzen01Chlosyne Janzen02Chlosyne Janzen02Chlosyne Janzen02Chlosyne Janzen02Chlosyne laciniaDHJ01Chlosyne laciniaDHJ01Chlosyne laciniaDHJ02

Chlosyne laciniaDHJ02Chlosyne laciniaDHJ02Chlosyne laciniaDHJ02Chlosyne laciniaDHJ02

Chlosyne gaudialisChlosyne erodyle

Pteronymia latillaDHJ01Pteronymia latillaDHJ01Pteronymia latillaDHJ01

Pteronymia latillaDHJ02Pteronymia latillaDHJ02Pteronymia latillaDHJ02Pteronymia latillaDHJ02Pteronymia latillaDHJ02

Pteronymia pictaDynamine mylittaDHJ01Dynamine mylittaDHJ01

Dynamine mylittaDHJ02Dynamine mylittaDHJ02Dynamine mylittaDHJ02Dynamine mylittaDHJ02

Dynamine mylittaDHJ02Dynamine sosthenes

Caligo atreusCaligo eurilochusCaligo telamoniusDHJ01

Caligo telamoniusDHJ01Caligo telamoniusDHJ01

Caligo telamoniusDHJ01Caligo telamoniusDHJ01Caligo telamoniusDHJ02Caligo telamoniusDHJ02Caligo telamoniusDHJ02Caligo telamoniusDHJ02Caligo telamoniusDHJ02

5 changes

Caligotelamonius(Felder & Felder 1862)

Dynamine mylitta(Cramer 1782)

Pteronymialatilla ( Hewitson[1855])

Chlosyne lacinia(Geyer 1837)

ChlosyneJanzen 1 & 2

Biblis hyperia(Cramer 1782)

Prepona laertes(Hubner [1811])

Memphis forreri(Godman & Salvin 1884)

Sideronemartesia(Cramer 1777)

Zaretis isidora(Cramer 1779)

Memphis moruus( Fabricius 1775)

Taygetisandromeda(Cramer 1779)

Adelpha zina( Hewitson 1867)

Adelphabasiloides(Bates 1865)

Figure 3: One of 200 most parsimonius cladograms inferred from barcode sequences showing 15 potential new Nymphalidae species. Converted to a phylogram to visualize number of nucleotide changes supporting the branch es.

S101778S101780

S103496S103497S103505S103850

S103656S103009

S102320S102321S103011

S101781S102322

S104088S104089S101784S102789S103014S104085S104338S104337S102312

S102308S103654

S102309S103140S103399S103507S103508S103846

S103397S103398S103509S103928

S103494S103510

S104095S103655

S103847S102310

S102311S102786S102787S102788S103143S103400S103492S103842

S103843S103844S103926

S102313S103493S103950

S104086S104087

S102314S102315S102316S102317S102318S102795S102796

S102797S103010S102319

Enyo1Enyo2

Manduca1Manduca2

S103401S103848

S104084S103495S103504S103849

S103141S103142S103490S103491S103841S104339

S103845S104094

S104340

10 changes

S101778S101780

S103496S103497S103850

S103505S103656

S103009S102320

S102321S103011

S101781S102322S104089S104088

S101784S102312S102789

S103014S104085

S104337S104338

S102310S102311S102786S102787S103143S103844S103926

S102788S103400S103492S103842S103843

S103141S103491S103490S103142

S104339S103841S103845S102313

S103950S103493

S104086S104087S102314S102315S102316S102317S102318

S102319S102795S102796S102797S103010

S103401S103848S104084

S103495S103504S103849

S104340S102308

S103654S103655S103847S103140S103397S103507S103509

S103508S103846S103928

S103398S103399

S103494S102309

S103510S104095

S104094

5 changes

Species discovery: testing nuclear gene regions

Cytochrome c oxidase I

10 changes 5 changes

Wingless protein

Rothschildia lebeau

Caio championi

Othorene purpurascens

Rothschildia lebeau

Rothschildia lebeau

Adeloneivaia jason

Citheronia bellavista

Eacles ormondei

Eacles imperialis

Eacles masoni

Adeloneivaia quadrilineata

Copaxa rufinans

Synonymizing species: test study Atteva

Dry Forest

Atteva punctella/ergatica

Rain Forest

Atteva punctella (Cramer, [1781])

Atteva ergatica (Walsingham, 1914)

One Species? Two Names?

Synonymizing species: test study Atteva

Next steps:

•Morphological examination•Comparison with A. ergatica holotype in BMNH



Species discovery

Deeper hierarchical relationships

Phylogenetic utility: questions

What is the phylogenetic information content of these sequences, and will these data contribute to a solid image of the tree of life? (Savolainen et al. 2005; Vogler & Monaghan 2006)

How would any phylogenetic reconstruction be affected by taxa sampling?(Savolainen et al. 2005; Tautz et al. 2003)

Phylogenetic utility: testing with ‘known’ phylogenies

Phylogeny Taxonomy

Blue Sister species

Yellow Sister species

Red Genus

Green Subfamily

Orange Family

If the COI dataset is useful for phylogenetic analysis then the barcode analysis must consistently recover ‘test’ clades hypothesized in independently derived

cladograms

Build COI cladogram and look for concordant branches

Phylogenetic utility: taxa addition

Test clades from literature:

Genus1Genus2

Few taxa:

5 terminals3 terminals

Increased taxa:

8 terminals7 terminals

If increased sampling of taxa is advantageous to the recovery of the tree of life then the addition of terminals should increase the recovery and support of ‘test’ clades

Summary of research goals

BIOLEP INVENTORY ENABLES THE FOLLOWING:

Testing the hypothesis that species can be identified using the barcoding technique

- Effect of different approaches: phenetic, character-based, likelihood

Investigating the usefulness of DNA barcodes in species discovery (and uncovering synonymy)

- Ability to produce corroborated robust species hypotheses tested through independent sources of data (nuclear genes)

- Explore different delimitation approaches within the framework of the phylogenetic species concept

Testing the phylogenetic utility of barcode characters

- Effect of increased taxa sampling

Acknowledgements

Laboratory Database & Analysis

Funding & Support

Stephanie Kirk, Lynne Oslach, Becky Cowling, Chris Grainger, Angela Holliss, Shana Hayter &

Luiqhong Lu

Greg Downs, Riadul Mannan & Sujeevan Ratnasingham

GuidanceMehrdad Hajibabaei, Dan

Janzen, Jean-Francois Landry, Rodolphe Rougerie,

Hebert Lab Grad Students, DNA Barcoding Discussion Group &

IB Science Communication Class

Gordon and Betty Moore Foundation and NSERC to PDN

Hebert, NSF and Wege Foundation to DH Janzen and

W Hallwachs

Questions

DNA barcoding of the Lepidoptera of Area de Conservacion Guanacaste, Costa Rica

Documents

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