Barcoding Anurans from Brazilian Atlantic Forest
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2 Barcoding Anurans from Brazilian Atlantic Forest Mariana L. Lyra, Célio F. B. Haddad & Ana Maria L. de Azeredo-Espin Capes/CNPq Prodoc nº /05-9 International Symposium on DNA Barcoding São Paulo Dezember 2009
3 Laboratório Genética Animal (UNICAMP) Profª Ana Maria L. Azeredo- Espin Molecular Markers mtdna + Laboratório de Herpetologia (UNESP-Rio Claro) Prof. Célio F. B. Haddad Biggest tissue and specimens collection of Anurans (Brazil) Pilot Project: Testing COI DNA Barcode in Anurans from Brazilian Atlantic Forest
4 Barcoding Anurans from Brazilian Atlantic Forest Mariana L. Lyra, Célio F. B. Haddad & Ana Maria L. de Azeredo-Espin Capes/CNPq Prodoc nº /05-9 International Symposium on DNA Barcoding São Paulo Dezember 2009
5 Phyllomedusa sp. Bolitoglossa paraensis Siphonops paulensis Amphibia sp. Anura sp. Caudata- 580 sp. Gymnophiona- 174 sp. Frost, Darrel R Amphibian Species of the World: an Online Reference. Version 5.3 (12 February, 2009). Electronic Database accessible at - American Museum of Natural History, New York, USA.
6 Phyllomedusa sp. Bolitoglossa paraensis Siphonops paulensis Amphibia sp. Anura sp. Caudata- 580 sp. Gymnophiona- 174 sp. Amphibia- 857 sp. Anura- 823 sp. Caudata- 2 sp. Gymnophiona- 33 sp. Frost, Darrel R Amphibian Species of the World: an Online Reference. Version 5.3 (12 February, 2009). Electronic Database accessible at - American Museum of Natural History, New York, USA.
7 Decline in amphibian populations worldwide National Geographic Brazil April 2009 e.g. Wake 1991, Alford & Richards 1999,Stuart et al. 2004, Vietes et al
8 Decline in amphibian populations worldwide National Geographic Brazil April Habitat Loss 2- Climate changes 3- Spread of microbial pathogens e.g. Wake 1991, Alford & Richards 1999,Stuart et al. 2004, Vietes et al
9 Decline in amphibian populations worldwide National Geographic Brazil April Habitat Loss 2- Climate changes 3- Spread of microbial pathogens e.g. Wake 1991, Alford & Richards 1999,Stuart et al. 2004, Vietes et al
10 Brazilian Atlantic Forest Original area 16% of the Brazilian territory Legend today original area Today: ~7-8% ~1,2-0,8% intact Data:MMA; INPE/ Fundação SOS mata atlântica, 2009
11 Brazilian Atlantic Forest Original area 16% of the Brazilian territory Legend today original area Today: ~7-8% ~1,2-0,8% intact Biodiversity hotspots 460 amphibian species ~90 endemic species-bioma Data:MMA; INPE/ Fundação SOS mata atlântica, 2009
12 Brazilian Atlantic Forest Original area 16% of the Brazilian territory Legend today original area Today: ~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
13 We need to know the species to conservate DNA barcoding has emerged at the forefront of efforts to accelerate the rates of taxonomic discovery
14 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
15 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)
16 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)
17 CFBH tissue collection (UNESP-Rio Claro) Aparasphenodon brunoi 58 anuran species Aplastodiscus callipygius 14 different families Xenohyla truncata Physalaemus atlanticus Proceratophrys appendiculata Flectonotus sp. Ceratophrys aurita Phylomedusa sp. Hypsiboas prasinus Arcovomer passarellii Images: Célio F B Haddad Ischnocnema sp. Brachycephalus epphipium
18 CFBH tissue collection (UNESP-Rio Claro) Aparasphenodon brunoi 58 anuran species Aplastodiscus callipygius 1 Caudata Xenohyla truncata Bolitoglossa paraensis Flectonotus sp. Phylomedusa sp. Hypsiboas prasinus Arcovomer passarellii Images: Célio F B Haddad Ischnocnema sp. Brachycephalus epphipium
19 CFBH tissue collection (UNESP-Rio Claro) Aparasphenodon brunoi 58 anuran species Aplastodiscus callipygius 1 Caudata 3 Gymnophiona Xenohyla truncata Siphonops paulensis Flectonotus sp. Phylomedusa sp. Hypsiboas prasinus Arcovomer passarellii Images: Célio F B Haddad Ischnocnema sp. Brachycephalus epphipium
20 Primers design 1- Previous works in Amphibian Barcode Vences et al (Amphibians from Madagascar) Smith et al (Holoartic amphibians) 2 33 complete COI sequences (GeneBank)
21 Primers design 1- Previous works in Amphibian Barcode Vences et al (Amphibians from Madagascar) Smith et al (Holoartic amphibians) 2 33 complete COI sequences (GeneBank) COI W A N C Y Barcode region S D COI AnF1 AnCOI-R AnR1
22 Primers design 1- Previous works in Amphibian Barcode Vences et al (Amphibians from Madagascar) Smith et al (Holoartic amphibians) 2 33 complete COI sequences (GeneBank) COI W A N C Y Barcode region S D COI AnW AnF1 AnCOI-R AnR1 AnD Nested or Semi-nested PCR- Museum specimens
23 Primers design 1- Previous works in Amphibian Barcode Vences et al (Amphibians from Madagascar) Smith et al (Holoartic amphibians) 2 33 complete COI sequences (GeneBank) COI W A N C Y Barcode region S D COI AnW AnF1 AnCOI-R AnR1 AnD 16S V L 16S An-16SF An-16SR
24 Primers design: Results COI and 16S - All species amplified 60 Works Fail AnF1+AnCOIR AnF1+AnR1 AW-AD 16S Barcode Region Nested
25 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)
26 Intra and interspecific diversity COI x 16S Number species Anura: 58 Number individuals: 170 ~3 ind/ sp Barcode region COI 16S
27 K2P distance Intraspecific diversity COI x 16S 0,16 COI 16S 0,12 0,08 0,04 0 COI: (K2P) 16S: (K2P)
28 K2P distance Intraspecific diversity COI x 16S 0,16 COI 16S 0,12 0,08 0,04 0 COI: (K2P) 16S: (K2P)
29 K2P distance Intraspecific diversity COI x 16S 0,16 COI 16S 0,12 0,08 0,04 0 Highly correlated with geographical distance
30 K2P distance Intraspecific diversity COI x 16S 0,16 COI 16S 0,12 0,08 0,04 0 Highly correlated with geographical distance Potential new species? look for other evidences
31 Nº species Nº species Interspecific diversity COI x 16S COI Interspecific variation 15-29% Intra Inter K2P distance 16 16S intra inter Interspecific variation 6-30% K2P distance
32 Nº species Nº species Interspecific diversity COI x 16S COI Interspecific variation 15-29% Candidate new species? Sc. catharinae Intra Inter Pseudopadicola cf. falcipes 16S intra inter Interspecific variation 6-30%
33 Nº species Nº species Interspecific diversity COI x 16S BUT Data came from very distinct anuran families Interspecific variation are probably over-estimated We need to include more congeneric species to understand intra and interspecific variation in anurans Intra Inter COI % variation intra inter 16S Can not suggest a threshold values to identify candidate species based on this data % variation
34 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)
35 Leptodactylus ocellatus (Linnaeus 1758) Célio F B Haddad 21 specimens
36 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 100 A238 L.ocellatus_ES A112 L.ocellatus_ES 98 A264 L.ocellatus_BA A235 L.ocellatus_SC A272 L.ocellatus_SP 100 A228 L.ocellatus_SP A253 L.ocellatus_SP A205 L.ocellatus_MG A219 L.ocellatus_SP 99 A222 L.ocellatus_MG A232 L.ocellatus_PR A221 L.ocellatus_PR A260 L.ocellatus_PR A267 L.ocellatus_RS 100 A236 L.ocellatus_RS Neighbor-joining/ K2P AND Gen. Mix.Yule-Coalescent model GMYC (Bayesian analysis)
37 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 98 A264 L.ocellatus_BA A235 L.ocellatus_SC A272 L.ocellatus_SP 100 A228 L.ocellatus_SP A253 L.ocellatus_SP A205 L.ocellatus_MG A219 L.ocellatus_SP 99 A222 L.ocellatus_MG A232 L.ocellatus_PR A221 L.ocellatus_PR A260 L.ocellatus_PR A267 L.ocellatus_RS 100 A236 L.ocellatus_RS
38 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 78 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 98 A264 L.ocellatus_BA A235 L.ocellatus_SC A272 L.ocellatus_SP 100 A228 L.ocellatus_SP A243 16S ES A242 16S BA A238 16S ES A271 16S RJ A237 16S RJ A112 16S ES A264 16S BA A272 16S SP A228 16S SP A235 16S SC A253 L.ocellatus_SP A205 L.ocellatus_MG A219 L.ocellatus_SP A253 16S SP A221 16S PR A219 16S SP A222 L.ocellatus_MG A232 L.ocellatus_PR A222 16S MG A232 16S PR A221 L.ocellatus_PR 90 A260 L.ocellatus_PR A267 L.ocellatus_RS 100 A236 L.ocellatus_RS A211 16S SC A205 16S MG A260 16S PR A267 16S RS A236 16S RS
39 Leptodactylus ocellatus: intra-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 98 A264 L.ocellatus_BA A235 L.ocellatus_SC A272 L.ocellatus_SP 100 A228 L.ocellatus_SP A253 L.ocellatus_SP K2P A205 L.ocellatus_MG A219 L.ocellatus_SP 99 A222 L.ocellatus_MG A232 L.ocellatus_PR A221 L.ocellatus_PR A260 L.ocellatus_PR A267 L.ocellatus_RS 100 A236 L.ocellatus_RS clade 1 clade 2: D=0.016 clade 3: D=0.004 clade 4: D=0.006 clade 5: D=0.011
40 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 A237 L.ocellatus_RJ A238 L.ocellatus_ES 78 A112 L.ocellatus_ES 98 A264 L.ocellatus_BA A235 L.ocellatus_SC D= A272 L.ocellatus_SP 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 A260 L.ocellatus_PR D=0.038 A267 L.ocellatus_RS 0.02 K2P 100 A236 L.ocellatus_RS
41 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 98 A264 L.ocellatus_BA A235 L.ocellatus_SC A272 L.ocellatus_SP 100 A228 L.ocellatus_SP A253 L.ocellatus_SP A205 L.ocellatus_MG A219 L.ocellatus_SP 99 A222 L.ocellatus_MG A232 L.ocellatus_PR A221 L.ocellatus_PR A260 L.ocellatus_PR A267 L.ocellatus_RS 100 A236 L.ocellatus_RS Strong correlated with geographical distribution
42 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 98 A264 L.ocellatus_BA A235 L.ocellatus_SC A272 L.ocellatus_SP 100 A228 L.ocellatus_SP A253 L.ocellatus_SP A205 L.ocellatus_MG A219 L.ocellatus_SP 99 A222 L.ocellatus_MG A232 L.ocellatus_PR A221 L.ocellatus_PR A260 L.ocellatus_PR A267 L.ocellatus_RS 100 A236 L.ocellatus_RS Bioacoustics and chromosomal differences coastal plain x plateau areas (Silva et al. 2000)
43 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 98 A264 L.ocellatus_BA A235 L.ocellatus_SC A272 L.ocellatus_SP 100 A228 L.ocellatus_SP A253 L.ocellatus_SP A205 L.ocellatus_MG A219 L.ocellatus_SP 99 A222 L.ocellatus_MG A232 L.ocellatus_PR A221 L.ocellatus_PR A260 L.ocellatus_PR A267 L.ocellatus_RS 100 A236 L.ocellatus_RS Bioacoustics and chromosomal differences coastal plain x plateau areas (Silva et al. 2000) Diversity is underestimated
44 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)
45 Scinax fuscovarius (Lutz, 1925) Foto: Raúl Maneyoro Foto: Lucas Grandinetti
46 Scinax fuscovarius: tree COI: NJ-K2P A258 S.fuscov SP 13 A200 S.fuscov SP 6 A234 S.fuscov SC 3 38 A220 S.fuscov SP A202 S.fuscov SP 1 A257 S.fuscov SP A254 S.fuscov SP 34 2 A146 S.fuscov SP 47 A227 S.fuscov SP A233 S.fuscov MG A51 S.fuscov SP A248 S.fuscov SP 54 A109 S.fuscov SP 15 A183 S.fuscov SP A201 S.fuscov SP 96 A250 S.fuscov SP A214 S.fuscov MS A230 S.fuscov SC A245 S.fuscov MS A212 S.fuscov SC 100 A179 S.fuscov Es 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
47 Scinax fuscovarius: geographic distribution COI: NJ-K2P A258 S.fuscov SP 13 A200 S.fuscov SP Diversity 6 A234 S.fuscov SC A220 S.fuscov SP 3 38 A202 S.fuscov SP 0 24% 1 A257 S.fuscov SP A254 S.fuscov SP 34 2 A146 S.fuscov SP 47 A227 S.fuscov SP A233 S.fuscov MG A51 S.fuscov SP A248 S.fuscov SP 54 A109 S.fuscov SP 15 A183 S.fuscov SP A201 S.fuscov SP 96 A250 S.fuscov SP A214 S.fuscov MS A230 S.fuscov SC A245 S.fuscov MS A212 S.fuscov SC 100 A179 S.fuscov Es 92 A204 S.fuscov GO 89 A203 S.fuscov GO A247 S.fuscov MT A226 S.fuscov MG A266 S.fuscov RS A241 S.fuscov SP Not correlated with geographic distance A197 S.fuscov SP 0.02
48 Scinax fuscovarius: collection COI: NJ-K2P A258 S.fuscov SP 13 A200 S.fuscov SP 6 A234 S.fuscov SC A220 S.fuscov SP A202 S.fuscov SP A257 S.fuscov SP Specimens collection: A254 S.fuscov SP A146 S.fuscov SP A227 S.fuscov SP Correction A233 S.fuscov MG A51 S.fuscov SP 38 A248 S.fuscov SP 54 A109 S.fuscov SP 15 A183 S.fuscov SP A201 S.fuscov SP A250 S.fuscov SP 96 A214 S.fuscov MS A197 A226 A241 A266 Scinax sp. Sc. eurydice Sc. cf. hayii Scinax sp. A230 S.fuscov SC A245 S.fuscov MS A212 S.fuscov SC 100 A179 S.fuscov Es 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
49 Scinax fuscovarius: synonymy COI: NJ-K2P 13 A258 S.fuscov SP A200 S.fuscov SP 6 A234 S.fuscov SC A220 S.fuscov SP A202 S.fuscov SP A257 S.fuscov SP A254 S.fuscov SP A146 S.fuscov SP A227 S.fuscov SP A233 S.fuscov MG A51 S.fuscov SP A248 S.fuscov SP A109 S.fuscov SP A183 S.fuscov SP A201 S.fuscov SP A250 S.fuscov SP A214 S.fuscov MS A230 S.fuscov SC A245 S.fuscov MS A212 S.fuscov SC A179 S.fuscov Es A204 S.fuscov GO 89 A203 S.fuscov GO Diversity: % A247 S.fuscov MT 0.02
50 Scinax fuscovarius: synonymy COI: NJ-K2P 13 A258 S.fuscov SP A200 S.fuscov SP 6 A234 S.fuscov SC A220 S.fuscov SP A202 S.fuscov SP A257 S.fuscov SP A254 S.fuscov SP A146 S.fuscov SP A227 S.fuscov SP A233 S.fuscov MG A51 S.fuscov SP A248 S.fuscov SP A109 S.fuscov SP A183 S.fuscov SP A201 S.fuscov SP A250 S.fuscov SP A214 S.fuscov MS A230 S.fuscov SC A245 S.fuscov MS A212 S.fuscov SC A179 S.fuscov Es A204 S.fuscov GO 89 A203 S.fuscov GO Diversity: % A247 S.fuscov MT 0.02
51 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 A202 S.fuscov SP A257 S.fuscov SP A254 S.fuscov SP A146 S.fuscov SP A227 S.fuscov SP A233 S.fuscov MG A51 S.fuscov SP A248 S.fuscov SP A109 S.fuscov SP A183 S.fuscov SP A201 S.fuscov SP A250 S.fuscov SP A214 S.fuscov MS A247 - Sc. megapodia(?) distribution Synonym revalidation? Look for other evidences A230 S.fuscov SC A245 S.fuscov MS A212 S.fuscov SC A179 S.fuscov Es A204 S.fuscov GO 89 A203 S.fuscov GO Diversity: % A247 S.fuscov MT 0.02
52 Scinax fuscovarius: criptic diversity? COI: NJ-K2P 13 A258 S.fuscov SP A200 S.fuscov SP 6 A234 S.fuscov SC A220 S.fuscov SP A202 S.fuscov SP A257 S.fuscov SP A254 S.fuscov SP A146 S.fuscov SP A227 S.fuscov SP A233 S.fuscov MG A51 S.fuscov SP A248 S.fuscov SP A109 S.fuscov SP A183 S.fuscov SP A201 S.fuscov SP A250 S.fuscov SP A214 S.fuscov MS A230 S.fuscov SC A245 S.fuscov MS 100 A212 S.fuscov SC Diversity ~2.6% A179 S.fuscov Es A204 S.fuscov GO A203 S.fuscov GO 0.02
53 Scinax fuscovarius: criptic diversity? COI: NJ-K2P 13 A258 S.fuscov SP A200 S.fuscov SP 6 A234 S.fuscov SC A220 S.fuscov SP A202 S.fuscov SP A257 S.fuscov SP A254 S.fuscov SP A146 S.fuscov SP A227 S.fuscov SP A233 S.fuscov MG A51 S.fuscov SP A248 S.fuscov SP A109 S.fuscov SP A183 S.fuscov SP A201 S.fuscov SP A250 S.fuscov SP A214 S.fuscov MS A230 S.fuscov SC A245 S.fuscov MS A212 S.fuscov SC A179 S.fuscov Es A204 S.fuscov GO A203 S.fuscov GO 0.02
54 Scinax fuscovarius: criptic diversity? COI: NJ-K2P 13 A258 S.fuscov SP A200 S.fuscov SP 6 A234 S.fuscov SC A220 S.fuscov SP A202 S.fuscov SP A257 S.fuscov SP A254 S.fuscov SP A146 S.fuscov SP A227 S.fuscov SP A233 S.fuscov MG A51 S.fuscov SP A248 S.fuscov SP A109 S.fuscov SP A183 S.fuscov SP A201 S.fuscov SP A250 S.fuscov SP A214 S.fuscov MS A230 S.fuscov SC A245 S.fuscov MS A212 S.fuscov SC A179 S.fuscov Es A204 S.fuscov GO A203 S.fuscov GO Geographic distribution of diversity? Look for other evidences
55 SUMARY AND CONCLUSIONS 1- Our primers for COI DNA Barcoding Anurans amplified all species sampled
56 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
57 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
58 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)
59 SUMARY AND CONCLUSIONS 5 - DNA barcode may help identify misclassification in collection (Biological collections certification)
60 SUMARY AND CONCLUSIONS 5 - DNA barcode may help identify misclassification in collection (Biological collections certification) 6- DNA barcode may help resolve problems with synonymy
61 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.
62 Obrigada Thank you Phylomedusa lemur Photo: ANAM CNPq Conselho Nacional de Desen volvimento Ci ent ífico e Te cno lógico MLL Fellowship Prodoc nº /05-9 CFBH collection- UNESP Laboratory projects UNICAMP
63 Barcoding Anurans from Brazilian Atlantic Forest Mariana L. Lyra, Célio F. B. Haddad & Ana Maria L. de Azeredo-Espin Capes/CNPq Prodoc nº /05-9 International Symposium on DNA Barcoding São Paulo Dezember 2009
64 GMYC Pons et al 2006 Syst.Biology 55: single Fontanero et al 2007 Plos Biology 5: single Monaghan et al 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)
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66 Single thresholds : 6 unidades coalescentes Outgroup
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68 t Coastal Ecosystems A m a z o n i a z o n e s t r a n s i t i o n C e r r a d o Coastal Ecosystems Pantanal A t l a n t i c F o r e s Southern Grasslands
69 Created a new account
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