DNA barcoding of South American mammals · The effort towards Large-scale DNA barcoding of...

The Biota-FAPESP International Symposium on DNA Barcoding – 2009

DNA barcoding of South American mammals

Eduardo Eizirik

Lab. Biologia Genômica e Molecular, PUCRS, Brasil

Instituto Pró-Carnívoros, Brasil

Laboratory of Genomic Diversity, NIH, EUA

Applications of DNA Barcoding in

Biodiversity Conservation

1. Gathering data on components of native biodiversity

1.1. Baseline data

1.2. Monitoring of biodiversity in impacted areas

2. Gathering data on threats to native biotas

- e.g. invasive species, pathogens, wildlife trafficking

3. Helping to enforce actions aimed at curbing threats to

biodiversity

- e.g. wildlife forensic analyses

Neotropical mammals

How DNA barcodes can help the study and conservation of

Neotropical mammals:

1. Improved reliability of surveys

- Academic-oriented (e.g. geographic range refinements)

- Environmental impact assessments

2. Ecological studies

- Habitat association

- Diet

- Parasite interactions

- Monitoring of species occurrence

3. Forensics and law enforcement applications

The effort towards Large-scale DNA barcoding of

Neotropical mammals

History:

Precursor:

2005 – Beginning of the Brazilian DNA barcoding network

- Large-scale inventory of Brazilian biodiversity

- Multi-center project designed in 2005 to boost taxonomic

research in Brazil using the DNA barcode concept as a catalyst to

integrate field collections, museum-based biodiversity research,

genome center networks and bioinformatics advances.

Large-scale inventorying of Brazilian biodiversity (2005) Sampling strategy

15 sites:

-10,000 samples/site:

- fish

- amphibians

- reptiles (incl. birds)

- mammals

- spiders

- Leguminosae

Large-scale inventorying of Brazilian biodiversity

6 museums

- Involved in coordinating collection trips, curation of sampled

specimens and their morphology-based analysis

14 Centers of Molecular Biodiversity:

- Network of genome centers and molecular biology laboratories

performing high-throughput sequencing of DNA barcodes from

150,000 samples;

Bioinformatics tools: online management of data collection,

analysis and integration (e.g. morphology, DNA barcodes,

geography)

Training activities: courses, workshops.


Neotropical mammals

History:

2007 – Regional CBOL meeting in Campinas, Brazil

- Mammals emerge as a CBOL-endorsed regional priority

for DNA barcoding.

- Several members of the 2005 network confirm

participation, while additional partner institutions join.


Neotropical mammals

History:

2007 – present

Activities:

1. Talks, symposia and/or planning meetings during subsequent

regional, national and international congresses:

- 2007, 2008 – Brazilian Congress of Genetics

- 2008 – Brazilian Congress of Zoology, Brazilian

Congress of Mammalogy, Regional Genetics Meeting (RS)

- 2009 – International Mammalogical Congress, Argentina

2. Funding proposals: CYTED (2007), PROSUL (2007, 2008).

PROSUL project proposal – 2008 (excerpt)

Goal:To consolidate an international network whose aim is generate, analyze and apply an integrated data base of DNA barcodes for South American mammals.

Official participants: 21 research institutions from six countries (Brazil, Uruguay, Chile, Bolivia, Peru and Venezuela).

- museum-based groups working on mammal systematics- molecular genetics laboratories

Proposed tasks:(i) Training of students from multiple countries in the use of molecular tools for biodiversity studies;(ii) Coordination meeting comprising group leaders;(iii) Reagent costs for the initial phase of data collection.

PROSUL 2008 participating institutionsBrazilPUCRS - Pontifícia Universidade Católica do Rio Grande do SulUFMG - Universidade Federal de Minas GeraisUFRGS - Universidade Federal do Rio Grande do SulUFES - Universidade Federal do Espírito SantoFZB/RS - Fundação Zoobotânica do Rio Grande do SulURI – Universidade Regional IntegradaFURB – Universidade Regional de BlumenauUFPR –Universidade Federal do ParanáUFRJ - Museu NacionalINCA - Instituto Nacional de CâncerFIOCRUZ - Fundação Oswaldo CruzUERJ - Universidade do Estado do Rio de JaneiroUSP - Instituto de BiociênciasUSP/ESALQ - Escola Superior de Agricultura Luiz de QueirozINPA - Instituto Nacional de Pesquisas da AmazôniaUFMS - Universidade Federal de Mato Grosso do SulUCB-DF - Universidade Católica de BrasíliaUFPA - Universidade Federal do ParáUFAM - Universidade Federal do Amazonas

PROSUL 2008 participating institutions

Uruguay

FC - Facultad de Ciencias (Enrique Lessa)

Chile

UC - Universidad de Chile (Angel Spotorno)

Venezuela

USB - Universidad Simon Bolivar (Marisol Aguilera)

Peru

UPCH - Universidad Peruana Cayetano Heredia (Jose R. Espinosa)

Bolivia

MNHN - Museo Nacional de Historia Natural (Julieta Vargas)

South American Network for Mammalian DNA barcoding

Regional Nodes

Brazil

1. Rio Grande do Sul – PUCRS/UFRGS

2. Espírito Santo – UFES

3. Minas Gerais – UFMG

4. Rio de Janeiro – INCA/UERJ

5. São Paulo - USP

6. Pará - UFPA

7. Goiás/Brasília - UFG

Uruguay

1. Universidad de la Republica

Carnivora

Xenarthra

Rodentia

Chiroptera

Small mammals

Xenarthra

Chiroptera

Xenarthra

Chiroptera

Rodentia

Rodentia

Primates

Primates

Species sampled so far:

Porto Alegre (PUCRS/UFRGS)Xenarthra – 5 spp.Carnivora – 23 spp. Next targets: Cetacea, Primates Rodentia – 43 spp.Cetartiodactyla – 4 spp.Chiroptera – 15 spp.Total: 90 spp (~ 600 specimens)

Belo Horizonte (UFMG)Xenarthra – 7 spp.Chiroptera – 35 spp. (> 200 specimens)

Espírito Santo (UFES)Rodentia, Didelphimorphia, Chiroptera – 85 spp. (259 specimens)

São Paulo (USP)Xenarthra – 6 spp. (149 specimens) [N. Moraes-Barros]

Next targets: Chiroptera, Rodentia [F. Martins]

bPgy35

bPgy06

Pgy05

Pgy02

Lycalopex gymnocercus

bPve05

bPve06

bPve04

Lycalopex vetulus

bCth208

bCth205

bCth164

bCthImperatrizMA

bCth185

bCth51

Cerdocyon thous

Lobo-guaraExtr1SCL

bCbr302II

Lobo-guara3

Cbr05

Chrysocyon brachyurus

Aau586

Aau683

Aau687

A.australis

Aau689L-159-H04

Arctocephalus australis

Arctocephalus forsteri

bPbr02

bPbr01Pteronura brasiliensis

Martes americana

Mam02Martes americana (Neártico)

Lcn1

Lontra canadensisLontra canadensis (Neártico)

bLlo30

bLlo06

bLlo15

bLlo29

bLlo31

Lontra longicaudis

bPon56

bP13Panthera onca

Panthera tigris

Puma concolor

Lco9

Lco10Leopardus colocolo

Oge38

bLge32

Oge63

Leopardus geoffroyi

bLti97

bLge01

bLti96

Leopardus tigrinus

100

100

100

100

70

99

98

82

92

99

91

74

86

99

100

100

100

100

81

100

62

99

83

98

78

99

99

60

76

81

100

0.02

Building a

carnivore barcode

database: COI

Brazil

Bolívia

Urugua

y

Leopardus tigrinus

Leopardus colocolo

Leopardus pardalis

L

.

g

e

o

f

f

r

o

y

i

bLge28| Leopardus geoffroyi





bLg68| Leopardus geoffroyi





blge55| Leopardus geoffroyi

bLti59| Leopardus tigrinus




Lco07| Leopardus colocolo

Lpa32| Leopardus pardalis

86

99

81

85

86

100

3934

32

46

52

98

58

0.02

bPve305| Lycalopex vetulus

Pve310| Lycalopex vetulus






culp1208| Lycalopex culpaeus




bPgy35| Lycalopex gymnocercus



s.001| Lycalopex sechurea



Dg19| Lycalopex griseus

Dg8coi| Lycalopex griseus

Dgr15| Lycalopex griseus



Dfu29| Lycalopex fulvipes

Dfu29.1| Lycalopex fulvipes

Df27| Lycalopex fulvipes


Df20| Lycalopex fulvipes


96

70

27

31

89

100

98

61

36

52

87

77

61

56

63

43

87

73

55

79

60

86

59

84

0.002

Lycalopex vetulus

Lycalopex culpaeus

Lycalopex

gymnocercus

Lycalopex sechurae

Lycalopex griseus

Lycalopex fulvipes

Evolutionary ecology of L. tigrinus and L. geoffroyi

Leopardus tigrinus

Leopardus geoffroyi

?

L. colocolo

Habitat use of L. tigrinus and L. geoffroyi in RS state

100 miles

N

Uruguay

Argentina

Atlantic Ocean

Gramineous SteppePark SteppeArboreal SteppeSteppical SavannahSemidecidual Stational ForestDecidual Stational ForestMixed Ombrophilous ForestDense Ombrophilous Forest

Pioneer FormationsEcological Tension Area

Dietary analysis of L. tigrinus e L. geoffroyi

using stomach contents of roadkilled animals

0

5

10

15

20

25

30

Akod

on sp

Calom

ys sp

Del

omys d

orsa

lis

Holo

chilu

s bra

silie

nsis

Mus

mus

culu

s

Olig

orym

ozys

sp

Ory

zom

ys sp

Oxym

ycteru

s sp

Rattu

s ra

ttus

Cav

ia sp

Mon

odelph

is sp

Col

umbifo

rmes

Cuc

ulifo

rmes

Gru

iform

es

Passe

riform

es

Tinam

iform

es

Sauria

Serpen

tes

Anur

a

Prey category

% o

f p

rey in

die

t

Leopardus tigrinus

Leopardus geoffroyi

Development of molecular tools for carnivore

species identification from faecal DNA

“Molecular scatology meets DNA barcoding”

Assessment of variability in mtDNA segments

Generation of large data bases of carnivore sequences

Development of molecular tools for carnivore

species identification from faecal DNA

0.02

bPon56 (fezes Panthera onca em álcool)

bPon55 (fezes de Panthera onca em DET)

bPon306 (fezes de Panthera onca)


bPon56 (fezes Panthera onca - 98 mg)

bPon32 (sangue de Panthera onca)

bPon56 (fezes de Panthera onca em DET)

P3-16 (pele de Panthera onca)


P3-2 (pêlos de Panthera onca)

bPon56 (fezes de Panthera onca - 48 mg)

bPon24 (sangue de Panthera onca)

P31-1 (pêlos de Panthera onca)

F2-636 (fezes de felino grande)

Puma concolor (sangue)

L.V.(fezes de felino grande)

F57-1C (fezes de felino grande)

F2-635 (fezes de felino grande)

Felis catus

100

99

Haag et al. 2009

Identification of carnivores using short DNA segments

Goal: global standardized

saystem for carnivore

identification.

Application in ecological,

biogeographic and forensic

studies.

Ongoing tests in multiple field

sites in South America

Brazil (RS, ES, MS, MT, TO,

MG,DF), Argentina.

P.B. Chaves et al., in prep.


Ongoing surveys and ecological

studies

Testing and optimization of protocols for African carnivores:

Partnership with CCF, Namibia.


car70R Reverse Complement

Car78

bFca57

bFca53

bFca52

bFca54

Car73

bFca59

bFca55

bFca58

bFca02

bFca56

NC 001700.1| Felis catus

Fma

Fma11

57

39

25

12

21

30

52

34

97

46

70

91

18


Car78

bFca57

bFca53

bFca52

bFca54

Car73

bFca59

bFca55

bFca58

bFca02bFca56


Fma

Fma11

Fni

Fni14

serval 3

serval 1

Lse

Lse18

AY598470.1|AY598470S01 Lynx canadensis

AH014070.1|SEG AY598470S Lynx canadensis

Car69

Cca21

Cca1

CarCaracal

Car49



car54copy

car47


Aju

NC 005212.1| Acinonyx jubatus

Car48

AY463959.1| Acinonyx jubatus


AF344830.1| Acinonyx jubatus




Car2

Ppa286

Car8ccfAndBrazil

CarLeop

Ple185

Ple153

car84copy Reverse Complement

lionF

Cfe1

car52

NC 008450.1| Neofelis nebulosa

Vci

bAbi

Ial2

Rme

Hja

Hpa1

Pcr

Hbr1

Hhy40

Ccr34

Ccr236

Phe

Pli

NC 001602.1| Halichoerus grypus

Zca

Oro

AY377297.1| Hydrurga leptonyx


NC 008425.1| Hydrurga leptonyx

Man68

AY377295.1| Ommatophoca rossii


Bal

Bas

AM711898.1| Spilogale putorius complete

bCar24

AB291076.1| Martes melampus

AB291075.1| Meles meles anakuma

AB291077.1| Enhydra lutris

Ist

Ssu

Car12

AY598506.1|AY598506S01 Alopex lagopus

AH014073.1|SEG AY598506S Alopex lagopus

CarBatEaredFox

carWildDog

B11and12and15

DQ480508.1| Canis lupus isolate 1 fro...

AY598494.1|AY598494S01 Canis lupus

DQ480502.1| Canis familiaris isolate ...

AH014072.1|SEG AY598494S Canis lupus




AY656738.1| Canis familiaris isolate ...

AY729880.1| Canis familiaris


NC 002008.4| Canis lupus familiaris





NC 008092.1| Canis lupus


car50copy

car85R Reverse ComplementAY656739.1| Canis familiaris isolate ...

NC 008093.1| Canis latrans

DQ480511.1| Canis latrans isolate 2 f...



Car79

car23 copy

Car53

Car81


Car71


Car57

Car56

Car11


Car82


Car9ccf

Car64

Car68

Car15

Car80


Car43

Car83

Car77

car44copy

car55copy

car62

car66

car65copy

car60copy

Car75

100

100

52

88

100

100

100

95

100

97

88

57

75

70

39

25

12

21

30

52

34

97

46

70

99

17

1

1

0

1

23

14

33

50

67

100

46

88

97

57

91

35

61

48

25

91

27

38

61

50

39

18

7

19

7

45

7

42

2

1

5

1

16

29

2

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3

7

27

0

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10

8

23

45

61

85

99

28

58

86

87

38

35

43

45

34

78

46

21

30

16

17

27

17

48

27

12

12

0,02

IUCN Cat Specialist Group

Car69

Cca21

Cca1

Caracal52

88

100


Car78

bFca57

bFca53

bFca52

bFca54

Car73

bFca59

bFca55

bFca58

bFca02bFca56


Fma

Fma11

Fni

Fni14

serval 3

serval 1

Lse

Lse18



Car69

Cca21

Cca1

CarCaracal

Car49



car54copy

car47


Aju


Car48







Car2

Ppa286

Car8ccfAndBrazil

CarLeop

Ple185

Ple153


lionF

Cfe1

car52


Vci

bAbi

Ial2

Rme

Hja

Hpa1

Pcr

Hbr1

Hhy40

Ccr34

Ccr236

Phe

Pli


Zca

Oro




Man68



Bal

Bas


bCar24




Ist

Ssu

Car12



CarBatEaredFox

carWildDog

B11and12and15


















car50copy






Car79

car23 copy

Car53

Car81


Car71


Car57

Car56

Car11


Car82


Car9ccf

Car64

Car68

Car15

Car80


Car43

Car83

Car77

car44copy

car55copy

car62

car66

car65copy

car60copy

Car75

100

100

52

88

100

100

100

95

100

97

88

57

75

70

39

25

12

21

30

52

34

97

46

70

99

17

1

1

0

1

23

14

33

50

67

100

46

88

97

57

91

35

61

48

25

91

27

38

61

50

39

18

7

19

7

45

7

42

2

1

5

1

16

29

2

0

3

7

27

0

0

0

1

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10

8

23

45

61

85

99

28

58

86

87

38

35

43

45

34

78

46

21

30

16

17

27

17

48

27

12

12

0,02


Car78

bFca57

bFca53

bFca52

bFca54

Car73

bFca59

bFca55

bFca58

bFca02bFca56


Fma

Fma11

Fni

Fni14

serval 3

serval 1

Lse

Lse18



Car69

Cca21

Cca1

CarCaracal

Car49



car54copy

car47


Aju


Car48







Car2

Ppa286

Car8ccfAndBrazil

CarLeop

Ple185

Ple153


lionF

Cfe1

car52


Vci

bAbi

Ial2

Rme

Hja

Hpa1

Pcr

Hbr1

Hhy40

Ccr34

Ccr236

Phe

Pli


Zca

Oro




Man68



Bal

Bas


bCar24




Ist

Ssu

Car12



CarBatEaredFox

carWildDog

B11and12and15


















car50copy






Car79

car23 copy

Car53

Car81


Car71


Car57

Car56

Car11


Car82


Car9ccf

Car64

Car68

Car15

Car80


Car43

Car83

Car77

car44copy

car55copy

car62

car66

car65copy

car60copy

Car75

100

100

52

88

100

100

100

95

100

97

88

57

75

70

39

25

12

21

30

52

34

97

46

70

99

17

1

1

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1

23

14

33

50

67

100

46

88

97

57

91

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61

48

25

91

27

38

61

50

39

18

7

19

7

45

7

42

2

1

5

1

16

29

2

0

3

7

27

0

0

0

1

1

1

0

10

8

23

45

61

85

99

28

58

86

87

38

35

43

45

34

78

46

21

30

16

17

27

17

48

27

12

12

0,02

Car49



car54copy

car47


Aju


Car48





car94R Reverse Complement17

1

1

0

1

23

14

33

50

46

97


Car78

bFca57

bFca53

bFca52

bFca54

Car73

bFca59

bFca55

bFca58

bFca02bFca56


Fma

Fma11

Fni

Fni14

serval 3

serval 1

Lse

Lse18



Car69

Cca21

Cca1

CarCaracal

Car49



car54copy

car47


Aju


Car48







Car2

Ppa286

Car8ccfAndBrazil

CarLeop

Ple185

Ple153


lionF

Cfe1

car52


Vci

bAbi

Ial2

Rme

Hja

Hpa1

Pcr

Hbr1

Hhy40

Ccr34

Ccr236

Phe

Pli


Zca

Oro




Man68



Bal

Bas


bCar24




Ist

Ssu

Car12



CarBatEaredFox

carWildDog

B11and12and15


















car50copy






Car79

car23 copy

Car53

Car81


Car71


Car57

Car56

Car11


Car82


Car9ccf

Car64

Car68

Car15

Car80


Car43

Car83

Car77

car44copy

car55copy

car62

car66

car65copy

car60copy

Car75

100

100

52

88

100

100

100

95

100

97

88

57

75

70

39

25

12

21

30

52

34

97

46

70

99

17

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23

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67

100

46

88

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57

91

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61

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91

27

38

61

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18

7

19

7

45

7

42

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1

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29

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7

27

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1

1

1

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10

8

23

45

61

85

99

28

58

86

87

38

35

43

45

34

78

46

21

30

16

17

27

17

48

27

12

12

0,02


Car2

Ppa286

Car8

CarLeop

Ple185

Ple153

car84

lionF75

99

88

57

35

61

48

91


Car78

bFca57

bFca53

bFca52

bFca54

Car73

bFca59

bFca55

bFca58

bFca02bFca56


Fma

Fma11

Fni

Fni14

serval 3

serval 1

Lse

Lse18



Car69

Cca21

Cca1

CarCaracal

Car49



car54copy

car47


Aju


Car48







Car2

Ppa286

Car8ccfAndBrazil

CarLeop

Ple185

Ple153


lionF

Cfe1

car52


Vci

bAbi

Ial2

Rme

Hja

Hpa1

Pcr

Hbr1

Hhy40

Ccr34

Ccr236

Phe

Pli


Zca

Oro




Man68



Bal

Bas


bCar24




Ist

Ssu

Car12



CarBatEaredFox

carWildDog

B11and12and15


















car50copy






Car79

car23 copy

Car53

Car81


Car71


Car57

Car56

Car11


Car82


Car9ccf

Car64

Car68

Car15

Car80


Car43

Car83

Car77

car44copy

car55copy

car62

car66

car65copy

car60copy

Car75

100

100

52

88

100

100

100

95

100

97

88

57

75

70

39

25

12

21

30

52

34

97

46

70

99

17

1

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1

23

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50

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100

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88

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57

91

35

61

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25

91

27

38

61

50

39

18

7

19

7

45

7

42

2

1

5

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16

29

2

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3

7

27

0

0

0

1

1

1

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10

8

23

45

61

85

99

28

58

86

87

38

35

43

45

34

78

46

21

30

16

17

27

17

48

27

12

12

0,02







car50

car85R






Car79

car23

Car53

Car81

car74R

Car71

car63R

Car57

Car56

Car11

car91R

Car82

car45R

Car9ccf

Car64

Car68

Car15

Car80

car90R

Car43

Car83

Car77

car44copy

car55copy

car62

car66

car65copy

car60copy

Car75

85

99

58

86

87

43

78

Current challenges for large-scale DNA barcoding

of Neotropical mammals:

1. Samples with no vouchers (especially medium-sized and

large species).

2. Museum vouchers with no tissue samples.

3. Unsampled species.

4. Poor geographic coverage for most species.

5. Taxonomically complex taxa (need for thorough revision).

6. Lack of manpower, lack of large-scale funding.

Students

Henrique Figueiró

Marina Favarini

Fernanda Valdez

Ezequiel Fabiano

Flávia Tirelli

Cristine Trinca

Camila Jaeger

Paulo B. Chaves

Vanessa G. Graeff

Gabriel Macedo

Support: CNPq, CAPES, PUCRS

ACKNOWLEDGEMENTS:

Carnivore scat ID projects

Fernanda Michalski (Pró-Carnívoros)

Laurie Marker (CCF)

Anne Schmidt-Küntzel (CCF)

ACKNOWLEDGEMENTS:

Y. Leite, L. P. Costa, V. Fagundes, A. Ditchfield (UFES)

F. R. Santos (UFMG)

L. Müller, P. C. Estrela, T. R. O. Freitas (UFRGS)

J. S. Morgante, N. Moraes-Barros, F. M. Martins (USP)

L. R. Oliveira (UNISINOS/GEMARS)

V. Valiati (UNISINOS)

S. L. Bonatto (PUCRS)

A.R. Percequillo (ESALQ/USP)

C. R. Bonvicino, H. Seuánez (INCA)

J.A. Oliveira (Museu Nacional)

Salvatore Siciliano (FIOCRUZ)

R. Collevatti (UFG)

H. Schneider, M.I. Sampaio (UFPA)

M. A. Jardim (FZB-RS)

F. B. Peters, L. F. Machado (ULBRA)

J. Koenemann (PUCRS – Uruguaiana)

M. Bordignon (UFMS)

A.C. M. de Oliveira (UFPA)

L. Geise (UERJ)

J.I.R. Porto (INPA), I. Farias (UFAM), L. Tchaika (UEMA)

F. Braga (Museu Capão da Imbuia - PR)

DNA barcoding of South American mammals · The effort towards Large-scale DNA barcoding of...

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