Barcoding Anurans from Brazilian Atlantic Forest

Mariana L. Lyra, Célio F. B. Haddad & Ana Maria L. de Azeredo-Espin [email protected] Capes/CNPq Prodoc nº 563.975/05-9

International Symposium on DNA Barcoding –São Paulo 03-04 Dezember 2009 Laboratório Genética Laboratório de Herpetologia Animal (UNICAMP) (UNESP-Rio Claro) Profª Ana Maria L. Azeredo- Prof. Célio F. B. Haddad Espin + Biggest tissue and specimens collection Molecular Markers of Anurans (Brazil) mtDNA

Pilot Project: Testing COI DNA Barcode in Anurans from Brazilian Atlantic Forest Barcoding Anurans from Brazilian Atlantic Forest

Mariana L. Lyra, Célio F. B. Haddad & Ana Maria L. de Azeredo-Espin [email protected] Capes/CNPq Prodoc nº 563.975/05-9

International Symposium on DNA Barcoding –São Paulo 03-04 Dezember 2009 Phyllomedusa sp. Bolitoglossa paraensis Siphonops paulensis

Amphibia- 6433 sp. Anura- 5679 sp. Caudata- 580 sp. Gymnophiona- 174 sp.

Frost, Darrel R. 2009. Amphibian Species of the World: an Online Reference. Version 5.3 (12 February, 2009). Electronic Database accessible at http://research.amnh.org/herpetology/amphibia/ - American Museum of Natural History, New York, USA. Phyllomedusa sp. Bolitoglossa paraensis Siphonops paulensis

Amphibia- 6433 sp. Anura- 5679 sp. Caudata- 580 sp. Gymnophiona- 174 sp.

Amphibia- 857 sp. Anura- 823 sp. Caudata- 2 sp. Gymnophiona- 33 sp.

Frost, Darrel R. 2009. Amphibian Species of the World: an Online Reference. Version 5.3 (12 February, 2009). Electronic Database accessible at http://research.amnh.org/herpetology/amphibia/ - American Museum of Natural History, New York, USA. Decline in amphibian populations worldwide

National Geographic Brazil – April 2009

e.g. Wake 1991, Alford & Richards 1999,Stuart et al. 2004, Vietes et al . 2009…. Decline in amphibian populations worldwide

National Geographic Brazil – April 2009

1- Habitat Loss 2- Climate changes 3- Spread of microbial pathogens

e.g. Wake 1991, Alford & Richards 1999,Stuart et al. 2004, Vietes et al . 2009…. Decline in amphibian populations worldwide

National Geographic Brazil – April 2009

1- Habitat Loss 2- Climate changes 3- Spread of microbial pathogens

e.g. Wake 1991, Alford & Richards 1999,Stuart et al. 2004, Vietes et al . 2009…. Brazilian Atlantic Forest

Original area

16% of the Brazilian territory

Legend today Today: original area ~7-8% ~1,2-0,8% intact

Data:MMA; INPE/ Fundação SOS mata atlântica, 2009 Brazilian Atlantic Forest

Original area

16% of the Brazilian territory

Legend today Today: original area ~7-8% ~1,2-0,8% intact

Biodiversity hotspots

460 amphibian species

~90 endemic species-Bioma

Data:MMA; INPE/ Fundação SOS mata atlântica, 2009 Brazilian Atlantic Forest

Original area

16% of the Brazilian territory

Legend today Today: original area ~7-8% ~1,2-0,8% intact

Biodiversity hotspots

460 amphibian species

~90 endemic species-Bioma

New species are described every year

How many are disappearing?

Data:MMA; INPE/ Fundação SOS mata atlântica, 2009 We need to know the species to conservate

DNA barcoding has emerged at the forefront of efforts to accelerate the rates of taxonomic discovery We need to know the species to conservate

DNA barcoding

has emerged at the forefront of efforts to accelerate the rates of taxonomic discovery

Amphibians COI DNA barcoding main problems:

1- the high variability of priming sites that hinder the application of universal primers

2- the observed distinct overlap of intraspecific and interspecific divergence values, which implies difficulties in the definition of threshold values to identify candidate species

Vences et al 2005; Smith et al. 2007 Objectives

1- Design new primers for standard COI amplifications in Anurans

2- Preliminary analysis of inter and intraspecific diversity in anurans from Brazilian atlantic forest: COI and 16S

3- Case study in Leptodactylus ocellatus (Anura: Leptodactylidae)

4- Case study in Scinax fuscovarius (Anura: Hylidae) Objectives

1- Design new primers for standard COI amplifications in Anurans

2- Preliminary analysis of inter and intraspecfiic diversity in anurans from Brazilian atlantic forest: COI and 16S

3- Case study in Leptodactylus ocellatus (Anura: Leptodactylidae)

4- Case study in Scinax fuscovarius (Anura: Hylidae) CFBH tissue collection (UNESP-Rio Claro) 1994-2009 58 anuran species Aparasphenodon brunoi

Aplastodiscus callipygius 14 different families

Physalaemus atlanticus Xenohyla truncata

Proceratophrys appendiculata

Ceratophrys aurita Phylomedusa sp. Flectonotus sp.

Hypsiboas prasinus Images: Célio F B Haddad Arcovomer passarellii Ischnocnema sp. Brachycephalus epphipium CFBH tissue collection (UNESP-Rio Claro) 1994-2009 58 anuran species Aparasphenodon brunoi

Aplastodiscus callipygius 1 Caudata

Xenohyla truncata

Bolitoglossa paraensis

Phylomedusa sp. Flectonotus sp.

Hypsiboas prasinus Images: Célio F B Haddad Arcovomer passarellii Ischnocnema sp. Brachycephalus epphipium CFBH tissue collection (UNESP-Rio Claro) 1994-2009 58 anuran species Aparasphenodon brunoi

Aplastodiscus callipygius 1 Caudata

3 Gymnophiona

Xenohyla truncata

Siphonops paulensis

Phylomedusa sp. Flectonotus sp.

Hypsiboas prasinus Images: Célio F B Haddad Arcovomer passarellii Ischnocnema sp. Brachycephalus epphipium Primers design

1- Previous works in Amphibian Barcode Vences et al. 2005 – (Amphibians from Madagascar) Smith et al. 2008 (Holoartic amphibians) 2 – 33 complete COI sequences (GeneBank) Primers design

1- Previous works in Amphibian Barcode Vences et al. 2005 – (Amphibians from Madagascar) Smith et al. 2008 (Holoartic amphibians) 2 – 33 complete COI sequences (GeneBank)

COI

Barcode region W A N C Y S D

COI

AnF1 AnCOI-R AnR1 Primers design

1- Previous works in Amphibian Barcode Vences et al. 2005 – (Amphibians from Madagascar) Smith et al. 2008 (Holoartic amphibians) 2 – 33 complete COI sequences (GeneBank)

COI

Barcode region W A N C Y S D

COI

AnW AnF1 AnCOI-R AnD AnR1 Nested or Semi-nested PCR- Museum specimens Primers design

1- Previous works in Amphibian Barcode Vences et al. 2005 – (Amphibians from Madagascar) Smith et al. 2008 (Holoartic amphibians) 2 – 33 complete COI sequences (GeneBank)

COI

Barcode region W A N C Y S D

COI

AnW AnF1 AnCOI-R AnD AnR1 16S V L 16S

An-16SF An-16SR Primers design: Results

COI and 16S - All species amplified

Works Fail 60

45

30

15

0 AnF1+AnCOIR AnF1+AnR1 AW-AD 16S

Barcode Region Nested Objectives

1- Design new primers for standard COI amplifications in Anurans

2- Preliminary analysis of inter and intraspecific diversity in anurans from Brazilian atlantic forest: COI and 16S

3- Case study in Leptodactylus ocellatus (Anura: Leptodactylidae)

4- Case study in Scinax fuscovarius (Anura: Hylidae) Intra and interspecific diversity – COI x 16S

Number species Anura: 58

Number individuals: 170 ~3 ind/ sp

Barcode region

COI

16S Intraspecific diversity – COI x 16S

COI 16S 0,16

0,12

0,08 K2P distance K2P

0,04

0

COI: 0.00-0.135 (K2P) 16S: 0.00-0.18 (K2P) Intraspecific diversity – COI x 16S

COI 16S 0,16

0,12

0,08 K2P distance K2P

0,04

0

COI: 0.00-0.135 (K2P) 16S: 0.00-0.18 (K2P) Intraspecific diversity – COI x 16S

COI 16S 0,16

0,12

0,08 K2P distance K2P

0,04

0

Highly correlated with geographical distance Intraspecific diversity – COI x 16S

COI 16S 0,16

0,12

0,08 K2P distance K2P

0,04

0

Highly correlated with geographical distance Potential new species? – look for other evidences Interspecific diversity – COI x 16S

20 18 Intra Inter 16 COI 14 12 Interspecific variation 10 15-29% 8

6 Nº speciesNº 4 2 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 K2P distance 16 14 intra inter 12

16S 10

8 Interspecific variation 6

6-30% speciesNº 4

2

0 0 0-1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 K2P distance Interspecific diversity – COI x 16S

20 18 Intra Inter 16 COI 14 12 Interspecific variation 10 15-29% 8

6 Nº speciesNº 4 2 0 Candidate new species? 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Sc. catharinae Pseudopadicola cf. falcipes 16 14 intra inter 12

16S 10

8 Interspecific variation 6

6-30% speciesNº 4

2

0 0 0-1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Interspecific diversity – COI x 16S

20 18 Intra Inter COI BUT 16 14 12 Data came from very distinct 10 anuran families 8

6 Nº speciesNº Interspecific variation are 4 probably over-estimated 2 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 We need to include more % variation 16 congeneric species to 14 16S understand intra and intra inter interspecific variation in 12 anurans 10 8

Can not suggest a threshold 6 Nº speciesNº values to identify candidate 4 species based on this data 2 0 0 0-1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 % variation Objectives

1- Design new primers for standard COI amplifications in Anurans

2- Preliminary analysis of inter and intraspecic diversity in anurans from Brazilian atlantic forest: COI and 16S

3- Case study in Leptodactylus ocellatus (Anura: Leptodactylidae)

4- Case study in Scinax fuscovarius (Anura: Hylidae) Leptodactylus ocellatus (Linnaeus 1758)

Célio F B Haddad

21 specimens http://www.iucnredlist.org/details/57151/0 Leptodactylus ocellatus: tree

A181 L.ocellatus_BA A243 L.ocellatus_ES A242 L.ocellatus_BA A271 L.ocellatus_RJ A37 L.ocellatus_SP 96 A237 L.ocellatus_RJ Neighbor-joining/ K2P 100 A238 L.ocellatus_ES AND A112 L.ocellatus_ES 78 Gen. Mix.Yule-Coalescent model – GMYC A264 L.ocellatus_BA 98 (Bayesian analysis) A235 L.ocellatus_SC A272 L.ocellatus_SP 100 A228 L.ocellatus_SP A253 L.ocellatus_SP

73 A205 L.ocellatus_MG A219 L.ocellatus_SP

99 A222 L.ocellatus_MG A232 L.ocellatus_PR A221 L.ocellatus_PR 90 100 A260 L.ocellatus_PR A267 L.ocellatus_RS

0.02 100 A236 L.ocellatus_RS Leptodactylus ocellatus: Clades

COI – NJ K2P

A181 L.ocellatus_BA A243 L.ocellatus_ES A242 L.ocellatus_BA A271 L.ocellatus_RJ A37 L.ocellatus_SP 96 A237 L.ocellatus_RJ 100 A238 L.ocellatus_ES A112 L.ocellatus_ES 78 98 A264 L.ocellatus_BA A235 L.ocellatus_SC A272 L.ocellatus_SP 100 A228 L.ocellatus_SP A253 L.ocellatus_SP

73 A205 L.ocellatus_MG A219 L.ocellatus_SP

99 A222 L.ocellatus_MG A232 L.ocellatus_PR A221 L.ocellatus_PR 90 100 A260 L.ocellatus_PR A267 L.ocellatus_RS

0.02 100 A236 L.ocellatus_RS Leptodactylus ocellatus: clades COI x 16S

COI – NJ K2P 16S – NJ K2P

A181 L.ocellatus_BA A181 16S BA A243 L.ocellatus_ES A037 16S SP A242 L.ocellatus_BA A243 16S ES A271 L.ocellatus_RJ A242 16S BA A37 L.ocellatus_SP A238 16S ES 96 A271 16S RJ A237 L.ocellatus_RJ A237 16S RJ 89 100 A238 L.ocellatus_ES A112 16S ES A112 L.ocellatus_ES 68 78 A264 16S BA 98 A264 L.ocellatus_BA A272 16S SP A235 L.ocellatus_SC A228 16S SP 100 A272 L.ocellatus_SP 100 A235 16S SC A228 L.ocellatus_SP A253 L.ocellatus_SP A253 16S SP 73 A205 L.ocellatus_MG A221 16S PR 88 A219 L.ocellatus_SP A219 16S SP 99 A222 L.ocellatus_MG A222 16S MG A232 L.ocellatus_PR A232 16S PR A221 L.ocellatus_PR A211 16S SC 90 100 A260 L.ocellatus_PR A205 16S MG A267 L.ocellatus_RS A260 16S PR 99 A267 16S RS 0.02 100 A236 L.ocellatus_RS A236 16S RS 0.02 Leptodactylus ocellatus: intra-clade diversity

A181 L.ocellatus_BA clade 1 A243 L.ocellatus_ES A242 L.ocellatus_BA A271 L.ocellatus_RJ clade 2: D=0.016 A37 L.ocellatus_SP 96 A237 L.ocellatus_RJ 100 A238 L.ocellatus_ES A112 L.ocellatus_ES 78 98 A264 L.ocellatus_BA A235 L.ocellatus_SC A272 L.ocellatus_SP 100 clade 3: D=0.004 A228 L.ocellatus_SP A253 L.ocellatus_SP

73 A205 L.ocellatus_MG A219 L.ocellatus_SP clade 4: D=0.006 99 A222 L.ocellatus_MG A232 L.ocellatus_PR A221 L.ocellatus_PR 90 100 A260 L.ocellatus_PR A267 L.ocellatus_RS clade 5: D=0.011 0.02 K2P 100 A236 L.ocellatus_RS Leptodactylus ocellatus: inter-clade diversity

A181 L.ocellatus_BA A243 L.ocellatus_ES A242 L.ocellatus_BA A271 L.ocellatus_RJ A37 L.ocellatus_SP 96 A237 L.ocellatus_RJ 100 A238 L.ocellatus_ES A112 L.ocellatus_ES 78 98 A264 L.ocellatus_BA A235 L.ocellatus_SC D=0.147 A272 L.ocellatus_SP 100 A228 L.ocellatus_SP A253 L.ocellatus_SP

73 A205 L.ocellatus_MG A219 L.ocellatus_SP

99 A222 L.ocellatus_MG A232 L.ocellatus_PR A221 L.ocellatus_PR D=0.038 90 100 A260 L.ocellatus_PR A267 L.ocellatus_RS 0.02 K2P 100 A236 L.ocellatus_RS Leptodactylus ocellatus: geographic distribution

A181 L.ocellatus_BA A243 L.ocellatus_ES A242 L.ocellatus_BA A271 L.ocellatus_RJ A37 L.ocellatus_SP 96 A237 L.ocellatus_RJ 100 A238 L.ocellatus_ES A112 L.ocellatus_ES 78 98 A264 L.ocellatus_BA A235 L.ocellatus_SC A272 L.ocellatus_SP 100 A228 L.ocellatus_SP A253 L.ocellatus_SP

73 A205 L.ocellatus_MG A219 L.ocellatus_SP

99 A222 L.ocellatus_MG A232 L.ocellatus_PR A221 L.ocellatus_PR 90 100 A260 L.ocellatus_PR A267 L.ocellatus_RS

0.02 100 A236 L.ocellatus_RS Strong correlated with geographical distribution Candidate new species?

A181 L.ocellatus_BA A243 L.ocellatus_ES A242 L.ocellatus_BA A271 L.ocellatus_RJ A37 L.ocellatus_SP 96 A237 L.ocellatus_RJ 100 A238 L.ocellatus_ES A112 L.ocellatus_ES 78 98 A264 L.ocellatus_BA Bioacoustics and chromosomal differences A235 L.ocellatus_SC – coastal plain x plateau areas A272 L.ocellatus_SP 100 A228 L.ocellatus_SP (Silva et al. 2000) A253 L.ocellatus_SP

73 A205 L.ocellatus_MG A219 L.ocellatus_SP

99 A222 L.ocellatus_MG A232 L.ocellatus_PR A221 L.ocellatus_PR 90 100 A260 L.ocellatus_PR A267 L.ocellatus_RS

0.02 100 A236 L.ocellatus_RS Candidate new species?

A181 L.ocellatus_BA A243 L.ocellatus_ES A242 L.ocellatus_BA A271 L.ocellatus_RJ A37 L.ocellatus_SP 96 A237 L.ocellatus_RJ 100 A238 L.ocellatus_ES A112 L.ocellatus_ES 78 98 A264 L.ocellatus_BA Bioacoustics and chromosomal differences A235 L.ocellatus_SC – coastal plain x plateau areas A272 L.ocellatus_SP 100 A228 L.ocellatus_SP (Silva et al. 2000) A253 L.ocellatus_SP

73 A205 L.ocellatus_MG A219 L.ocellatus_SP

99 A222 L.ocellatus_MG A232 L.ocellatus_PR A221 L.ocellatus_PR 90 Diversity is underestimated 100 A260 L.ocellatus_PR A267 L.ocellatus_RS

0.02 100 A236 L.ocellatus_RS Objectives

1- Design new primers for standard COI amplifications in Anurans

2- Preliminary analysis of inter and intraspecic diversity in anurans from Brazilian atlantic forest: COI and 16S

3- Case study in Leptodactylus ocellatus (Anura: Leptodactylidae)

4- Case study in Scinax fuscovarius (Anura: Hylidae) Scinax fuscovarius (Lutz, 1925)

Foto: Raúl Maneyoro

Foto: Lucas Grandinetti http://www.iucnredlist.org/apps/redlist/details/55958/0 Scinax fuscovarius: tree

COI: NJ-K2P A258 S.fuscov SP 13 A200 S.fuscov SP 6 A234 S.fuscov SC

3 A220 S.fuscov SP 38 A202 S.fuscov SP 1 A257 S.fuscov SP A254 S.fuscov SP

342 A146 S.fuscov SP 47 A227 S.fuscov SP A233 S.fuscov MG 33 11 A51 S.fuscov SP 38 A248 S.fuscov SP

54 A109 S.fuscov SP

15 A183 S.fuscov SP 63 49 A201 S.fuscov SP A250 S.fuscov SP 96 A214 S.fuscov MS A230 S.fuscov SC

91 96 A245 S.fuscov MS 100 A212 S.fuscov SC A179 S.fuscov Es 100 92 A204 S.fuscov GO 89 A203 S.fuscov GO A247 S.fuscov MT A226 S.fuscov MG

80 A266 S.fuscov RS 67 A241 S.fuscov SP A197 S.fuscov SP

0.02 Scinax fuscovarius: geographic distribution

COI: NJ-K2P A258 S.fuscov SP 13 Diversity A200 S.fuscov SP 6 A234 S.fuscov SC A220 S.fuscov SP 3 0 – 24% 38 A202 S.fuscov SP 1 A257 S.fuscov SP A254 S.fuscov SP

342 A146 S.fuscov SP 47 A227 S.fuscov SP A233 S.fuscov MG 33 11 A51 S.fuscov SP 38 A248 S.fuscov SP

54 A109 S.fuscov SP

15 A183 S.fuscov SP 63 49 A201 S.fuscov SP A250 S.fuscov SP 96 A214 S.fuscov MS A230 S.fuscov SC

91 96 A245 S.fuscov MS 100 A212 S.fuscov SC A179 S.fuscov Es 100 92 A204 S.fuscov GO 89 A203 S.fuscov GO A247 S.fuscov MT A226 S.fuscov MG

80 A266 S.fuscov RS 67 A241 S.fuscov SP Not correlated with geographic distance A197 S.fuscov SP

0.02 Scinax fuscovarius: collection

COI: NJ-K2P A258 S.fuscov SP 13 A200 S.fuscov SP 6 A234 S.fuscov SC

3 A220 S.fuscov SP 38 A202 S.fuscov SP 1 A257 S.fuscov SP Specimens collection: A254 S.fuscov SP 342 A146 S.fuscov SP Correction 47 A227 S.fuscov SP A233 S.fuscov MG 33 11 A51 S.fuscov SP A197 Scinax sp. 38 A248 S.fuscov SP Sc. eurydice 54 A109 S.fuscov SP A226 15 A183 S.fuscov SP A241 Sc. cf. hayii 63 49 A201 S.fuscov SP A250 S.fuscov SP A266 Scinax sp. 96 A214 S.fuscov MS A230 S.fuscov SC

91 96 A245 S.fuscov MS 100 A212 S.fuscov SC A179 S.fuscov Es 100 92 A204 S.fuscov GO 89 A203 S.fuscov GO A247 S.fuscov MT A226 S.fuscov MG

80 A266 S.fuscov RS 67 A241 S.fuscov SP A197 S.fuscov SP

0.02 Scinax fuscovarius: synonymy

COI: NJ-K2P

A258 S.fuscov SP 13 A200 S.fuscov SP

6 A234 S.fuscov SC

A220 S.fuscov SP 3 38 A202 S.fuscov SP

1 A257 S.fuscov SP

A254 S.fuscov SP

A146 S.fuscov SP 342 47 A227 S.fuscov SP

A233 S.fuscov MG

33 A51 S.fuscov SP 11 38 A248 S.fuscov SP

A109 S.fuscov SP 54 A183 S.fuscov SP 15 63 49 A201 S.fuscov SP

A250 S.fuscov SP 96 A214 S.fuscov MS

A230 S.fuscov SC

91 A245 S.fuscov MS 96 Diversity: 100 A212 S.fuscov SC

A179 S.fuscov Es 3.6 - 4.4% 100 A204 S.fuscov GO 92 89 A203 S.fuscov GO

A247 S.fuscov MT

0.02 Scinax fuscovarius: synonymy

COI: NJ-K2P

A258 S.fuscov SP 13 A200 S.fuscov SP

6 A234 S.fuscov SC

A220 S.fuscov SP 3 38 A202 S.fuscov SP

1 A257 S.fuscov SP

A254 S.fuscov SP

A146 S.fuscov SP 342 47 A227 S.fuscov SP

A233 S.fuscov MG

33 A51 S.fuscov SP 11 38 A248 S.fuscov SP

A109 S.fuscov SP 54 A183 S.fuscov SP 15 63 49 A201 S.fuscov SP

A250 S.fuscov SP 96 A214 S.fuscov MS

A230 S.fuscov SC

91 A245 S.fuscov MS 96 Diversity: 100 A212 S.fuscov SC

A179 S.fuscov Es 3.6 - 4.4% 100 A204 S.fuscov GO 92 89 A203 S.fuscov GO

A247 S.fuscov MT

0.02 Scinax fuscovarius: synonymy

COI: NJ-K2P

A258 S.fuscov SP 13 A200 S.fuscov SP

6 A234 S.fuscov SC Scinax megapodia x Scinax fuscovarius

A220 S.fuscov SP 3 38 A202 S.fuscov SP

1 A257 S.fuscov SP

A254 S.fuscov SP

A146 S.fuscov SP A247 - Sc. megapodia(?) distribution 342 47 A227 S.fuscov SP A233 S.fuscov MG Synonym revalidation? 33 A51 S.fuscov SP 11 38 A248 S.fuscov SP

A109 S.fuscov SP 54 Look for other evidences A183 S.fuscov SP 15 63 49 A201 S.fuscov SP

A250 S.fuscov SP 96 A214 S.fuscov MS

A230 S.fuscov SC

91 A245 S.fuscov MS 96 Diversity: 100 A212 S.fuscov SC

A179 S.fuscov Es 3.6 - 4.4% 100 A204 S.fuscov GO 92 89 A203 S.fuscov GO

A247 S.fuscov MT

0.02 Scinax fuscovarius: criptic diversity?

COI: NJ-K2P

A258 S.fuscov SP 13 A200 S.fuscov SP

6 A234 S.fuscov SC

A220 S.fuscov SP 3 38 A202 S.fuscov SP

1 A257 S.fuscov SP

A254 S.fuscov SP

A146 S.fuscov SP 342 47 A227 S.fuscov SP

A233 S.fuscov MG

33 A51 S.fuscov SP 11 38 A248 S.fuscov SP

A109 S.fuscov SP 54 A183 S.fuscov SP 15 63 49 A201 S.fuscov SP

A250 S.fuscov SP 96 A214 S.fuscov MS Diversity A230 S.fuscov SC ~2.6% 91 A245 S.fuscov MS 96 100 A212 S.fuscov SC

A179 S.fuscov Es

A204 S.fuscov GO 92 89 A203 S.fuscov GO

0.02 Scinax fuscovarius: criptic diversity?

COI: NJ-K2P

A258 S.fuscov SP 13 A200 S.fuscov SP

6 A234 S.fuscov SC

A220 S.fuscov SP 3 38 A202 S.fuscov SP

1 A257 S.fuscov SP

A254 S.fuscov SP

A146 S.fuscov SP 342 47 A227 S.fuscov SP

A233 S.fuscov MG

33 A51 S.fuscov SP 11 38 A248 S.fuscov SP

A109 S.fuscov SP 54 A183 S.fuscov SP 15 63 49 A201 S.fuscov SP

A250 S.fuscov SP 96 A214 S.fuscov MS

A230 S.fuscov SC

91 A245 S.fuscov MS 96 100 A212 S.fuscov SC

A179 S.fuscov Es

A204 S.fuscov GO 92 89 A203 S.fuscov GO

0.02 Scinax fuscovarius: criptic diversity?

COI: NJ-K2P

A258 S.fuscov SP 13 A200 S.fuscov SP

6 A234 S.fuscov SC

A220 S.fuscov SP 3 38 A202 S.fuscov SP

1 A257 S.fuscov SP

A254 S.fuscov SP

A146 S.fuscov SP 342 47 A227 S.fuscov SP

A233 S.fuscov MG

33 A51 S.fuscov SP 11 38 A248 S.fuscov SP

A109 S.fuscov SP 54 A183 S.fuscov SP 15 63 49 A201 S.fuscov SP

A250 S.fuscov SP 96 A214 S.fuscov MS

A230 S.fuscov SC

91 A245 S.fuscov MS 96 100 A212 S.fuscov SC

A179 S.fuscov Es

A204 S.fuscov GO 92 89 A203 S.fuscov GO Geographic distribution of diversity? 0.02 Look for other evidences SUMARY AND CONCLUSIONS

1- Our primers for COI DNA Barcoding Anurans amplified all species sampled SUMARY AND CONCLUSIONS

1- Our primers for COI DNA Barcoding Anurans amplified all species sampled

2- Although both COI and 16S rRNA have a great potential for DNA barcoding amphibians, we strongly suggest a large-scale effort to barcode the amphibians using COI SUMARY AND CONCLUSIONS

1- Our primers for COI DNA Barcoding Anurans amplified all species sampled

2- Although both COI and 16S rRNA have a great potential for DNA barcoding amphibians, we strongly suggest a large-scale effort to barcode the amphibians using COI

3- Anuran diversity in Brazil is underestimated, and COI DNA barcodes may help flag potential new species SUMARY AND CONCLUSIONS

1- Our primers for COI DNA Barcoding Anurans amplified all species sampled

2- Although both COI and 16S rRNA have a great potential for DNA barcoding amphibians, we strongly suggest a large-scale effort to barcode the amphibians using COI

3- Anuran diversity in Brazil is underestimated, and COI DNA barcodes may help flag potential new species

4- DNA barcodes may help to accelerate taxonomic revision (bioacustics and morpho-anatomy) SUMARY AND CONCLUSIONS

5 - DNA barcode may help identify misclassification in collection (Biological collections certification) SUMARY AND CONCLUSIONS

5 - DNA barcode may help identify misclassification in collection (Biological collections certification)

6- DNA barcode may help resolve problems with synonymy SUMARY AND CONCLUSIONS

5 - DNA barcode may help identify misclassification in collection (Biological collections certification)

6- DNA barcode may help resolve problems with synonymy

7- The use of DNA barcode will help make a better estimative of amphibian species richness and may yield important findings for the knowledge and conservation of these species. Obrigada

Thank you

Phylomedusa lemur – CNPq Photo: ANAM Conselho Nacional de Desen volvimento Ci entífico e Tecnológico MLL Fellowship Prodoc nº 563.975/05-9

CFBH collection- UNESP Laboratory projects UNICAMP Barcoding Anurans from Brazilian Atlantic Forest

Mariana L. Lyra, Célio F. B. Haddad & Ana Maria L. de Azeredo-Espin [email protected] Capes/CNPq Prodoc nº 563.975/05-9

International Symposium on DNA Barcoding –São Paulo 03-04 Dezember 2009 GMYC

Pons et al 2006 – Syst.Biology 55: 595-609 – “single” Fontanero et al 2007 – Plos Biology 5: 914-921 – “single” Monaghan et al 2009 - Syst.Biology – advance access publ jul 2009 – “multiple”

“General Mixed Yule-Coalescent model – GMYC”

Identificar limites de “espécies/grupos” baseado nos ramos de uma árvore que contém espécies e populações. Processos coalescentes neutros (pops) X especiação

Ultrametric tree - MrBayes 3.2

Analise no R (split, ape, gee)

“Single thresholds”: 6 unidades coalescentes

Outgroup “Multiple thresholds”: 7 unidades coalescentes

Outgroup Coastal Ecosystems

e s A m a z o n i a n z o

n i o t i s n a r t

C e r r a d o Coastal Ecosystems

t

s

Pantanal e

r o F c t i n a l t

A

Southern Grasslands Created a new account

“ DNA Barcoding Anuran from Brazilian of Atlantic forest” Fellowship: Capes/CNPqProdoc nº 563.975/05-9

Order: Anura 15 families

76 species

Order: Gymnophiona 1 family

3 species

Order: Caudata 1 family

1 species

Total: 80 sp.