Success of DNA barcoding in distinguishing sister species of ...

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

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:

Barcoding sister-species of birds

Multigene Multigene phylogeniesphylogenies

Sister-species Sister-species selectionselection

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

COI alignmentCOI alignment

DNA DNA extractionextraction

Amplification and Amplification and sequencing of COIsequencing of COI

GenBank GenBank and BOLD and BOLD

searchsearch

Calculate COI Calculate COI corrected corrected

distances distances

Map COI of Map COI of sister-sister-

species in species in multigene multigene phylogenyphylogeny

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 bpDistances

Multigene phylogeny of shanks Bayesian analysis12S and 16S rDNAs, ATPase8, ATPase6, COIII, ND1, tRNA-ISO, ND2, ND5, Cytb: 7664 bp. Pereira and Baker 2005 Condor 107.

COI:651 bpDistances


Multigene phylogeny of Calidridine sandpipers COI:~763 bpDistances

Bayesian analysisRAG1 + mtDNA: 12,143 bp. Baker et al. In prep.

Multigene phylogeny of penguins COI:~615 bpDistances Bayesian analysis

RAG1, 12S, 16S, COI, Cytb: 5571 bp.

Pereira and Baker et al (2006) Proc. Biol. Sci

273.


Multigene phylogeny of kiwis

COI:~650 bpDistances

Maximum LikelihoodATPAse 6, ATPase 8, CR, Cytb: 2267 bp.

Burbidge et al (2003) Conservation

Genetics 4.

* Bootstrap proportion = 100%

Multigene phylogeny of Pteroglossus toucans COI:~615 bpDistances

Bayesian analysisATPase, COI, Cytb: 2168 bp.

Eberhard and Bermingham (2005) MPE: 288-304


Multigene phylogeny of Woodpeckers

Bayesian analysisCOI, Cytb: 2580 bp.

Moore et al (2006) Biol. J. Linn. Soc.87


COI:~660 bpDistances

Sister-species barcode comparisons

Do many genes improve on COI barcoding of sister-species in 7 clades of birds?


Sister-species Sister-species selectionselection



COI alignment of COI alignment of well sampled well sampled species species DNA DNA

extractionextraction Amplification and Amplification and sequencing of COIsequencing of COI

GenBank GenBank and BOLD and BOLD

searchsearch

Calculate COI Calculate COI distances and NJ distances and NJ topologies: K2Ptopologies: K2P

Test barcode in

COI vs multigenes

Success in barcoding sister-species of birds

Sample of 812 sequences from 95 sister-species pairs

2 Clusters 1 Cluster

COI 93.7% (89) 6.3%(6)

Multigene 93.7% (89) 6.3%(6)

Of the 6 “failures” 4 were known to be from hybridization

Conclusion: No improvement using more genes

Intraspecific versus among sister-species variation

Sample of 812 sequences from 95 sister-species pairs.

All Birds

0

10

20

30

40

50

60

70

80

0.5 1 2 3 4 5 6 7 8 9 10

Percent Divergence (Kimura 2P)

Fre

qu

en

cy

Intraspecific

Sister-species

Conclusion: This is a potential problem for barcoding sister-species?

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)COI (651 bp)Multigene 3220 bp)Multigene 3220 bp)

New species of Little terns?

1.5%

1.3%

http://www.birdguides.com

Large-bill form

Small-bill form

1.7%

1.8%

New species of Gull-billed terns?

Gull-billed Tern

Within-species variation in Tringid sandpipers

Intra-specific



Neighbour Joining (model Kimura-2P) Node support: Posterior Prob. Bayesian analysis.

COI (623 bp)COI (623 bp)Multigene (7664 bp)Multigene (7664 bp)

*

Intra-specific



Within-species variation in penguins

PenguinsCOI (bp)COI (bp)Multigene (bp)Multigene (bp)

Neighbour Joining (model Kimura-2P)Node support: Posterior Prob. Bayesian analysis.

2- White-Flippered PenguinEudyptula m. albosignata

(J.N. Davis and J.Busby)

3-Eudyptula m. novaehollandiae


1-Little PenguinEudyptula m. minor


a,b

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%

New species of kiwis

COI 650 bpCOI 650 bpMultigene phylogenyMultigene phylogeny

Threshold for sister-species

Terns

0

5

10

15

20

25

0.5 1 2 3 4 5 6 7 8 9 10 15 20 30 40 50 60 70 80 90 100

Distances (TN+I+G)

Freq

uenc

y

Penguins

0

2

4

6

8

10

12

14

16

18

0.5 1 2 3 4 5 6 7 8 9 10 15 20 30 40 50 60 70 80 90 100

Distances GTR+I+G

Freq

uenc

y

Tringid sandpipers

0

2

4

6

8

10

12

0.5 1 2 3 4 5 6 7 8 9 10 15 20 30 40 50 60 70 80 90 100

Distances (GTR+I+G)

Fre

qu

ency

sister species

intraspecific

Toucans

0

2

4

6

8

10

12

0.5 1 2 3 4 5 6 7 8 9 10 15 20 30 40 50 60 70 80 90 100

Distances (GTR+I+G)

Fre

qu

ency

sister species

intraspecific

Excluding putative new species and hybridizing species

Calidridine sandpipers

0

2

4

6

8

10

12

14

16

0.5 1 2 3 4 5 6 8 9 10 11

Percent Divergence

Fre

qu

en

cy

Intraspecific

Sister-species


0

2

4

6

8

10

12

14

16

0.5 1 2 3 4 5 6 7 8 9 10 15 20 30 40 50 60 70 80 90 100

Fre

qu

ency

Distance (GTR+I+G)

Woodpeckers

intraspecif ic

sister-species

Conclusion: Sister-species and intraspecific variation overlap slightly around 1% level


COI barcodes vs time of divergence of species

Estimation of Estimation of divergence divergence times if not times if not available available

with with multidivtimemultidivtime




COI alignmentCOI alignment

COI distances vs COI distances vs divergence timesdivergence times

distances x time

0

2

4

6

8

10

12

14

16

18

0 5 10 15 20 25

divergence times

dis

tan

ce

s T

N+

g+

I

Model choiceModel choice

Species barcode level varies with time since speciation

0

2

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6

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14

16

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0 5 10 15 20 25

Div

erg

ence

Tim

es (

MY

)

Distances (GTR+I+G)

Sister-species Distances

Penguins

Curassous

Terns

Shanks

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

• 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

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.

• 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.

http://www.BOLNET.ca/

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