Post on 27-Sep-2020
Barcoding Anurans from Brazilian Atlantic Forest
Mariana L. Lyra, Célio F. B. Haddad & Ana Maria L. de Azeredo-Espin
marilyra@unicamp.br
Capes/CNPq Prodoc nº 563.975/05-9
International Symposium on DNA Barcoding –São Paulo 03-04 Dezember 2009
Laboratório de Herpetologia (UNESP-Rio Claro)
Prof. Célio F. B. Haddad
Biggest tissue andspecimens collection
of Anurans (Brazil)
Laboratório Genética Animal (UNICAMP)
Profª Ana Maria L. Azeredo-Espin
Molecular MarkersmtDNA
+
Pilot Project: Testing COI DNA Barcode in Anurans fromBrazilian Atlantic Forest
Barcoding Anurans from Brazilian Atlantic Forest
Mariana L. Lyra, Célio F. B. Haddad & Ana Maria L. de Azeredo-Espin
marilyra@unicamp.br
Capes/CNPq Prodoc nº 563.975/05-9
International Symposium on DNA Barcoding –São Paulo 03-04 Dezember 2009
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.
Siphonops paulensisBolitoglossa paraensisPhyllomedusa sp.
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.
Amphibia- 857 sp.
Anura- 823 sp. Caudata- 2 sp. Gymnophiona- 33 sp.
Siphonops paulensisBolitoglossa paraensisPhyllomedusa sp.
e.g. Wake 1991, Alford & Richards 1999,Stuart et al. 2004, Vietes et al . 2009….
National Geographic Brazil – April 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….
Decline in amphibian populations worldwide
Original area
16% of the Brazilian territory
Today:
~7-8%
~1,2-0,8% intact
Brazilian Atlantic Forest
Data:MMA; INPE/ Fundação SOS mata atlântica, 2009
today
original area
Legend
Biodiversity hotspots
Brazilian Atlantic Forest
Data:MMA; INPE/ Fundação SOS mata atlântica, 2009
today
original area
Legend
460 amphibian species
~90 endemic species-Bioma
Original area
16% of the Brazilian territory
Today:
~7-8%
~1,2-0,8% intact
Data:MMA; INPE/ Fundação SOS mata atlântica, 2009
today
original area
Legend
New species are described
every year
How many are
disappearing?
Biodiversity hotspots
Brazilian Atlantic Forest
460 amphibian species
~90 endemic species-Bioma
Original area
16% of the Brazilian territory
Today:
~7-8%
~1,2-0,8% intact
We need to know the species to conservate
DNA barcoding
has emerged at the forefront of efforts to accelerate the rates of taxonomic discovery
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
We need to know the species to conservate
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
14 different families
Images: Célio F B Haddad
Aparasphenodon brunoi
Aplastodiscus callipygius
Xenohyla truncataPhysalaemus atlanticus
Phylomedusa sp.Ceratophrys aurita
Arcovomer passarellii
Flectonotus sp.
Brachycephalus epphipiumIschnocnema sp.
Hypsiboas prasinus
Proceratophrys appendiculata
CFBH tissue collection
(UNESP-Rio Claro)
1994-2009
58 anuran species
1 Caudata
Images: Célio F B Haddad
Bolitoglossa paraensis
Aparasphenodon brunoi
Aplastodiscus callipygius
Xenohyla truncata
Phylomedusa sp.
Arcovomer passarellii
Flectonotus sp.
Brachycephalus epphipiumIschnocnema sp.
Hypsiboas prasinus
3 Gymnophiona
CFBH tissue collection
(UNESP-Rio Claro)
1994-2009
58 anuran species
1 Caudata
Images: Célio F B Haddad
Siphonops paulensis
Aparasphenodon brunoi
Aplastodiscus callipygius
Xenohyla truncata
Phylomedusa sp.
Arcovomer passarellii
Flectonotus sp.
Brachycephalus epphipiumIschnocnema sp.
Hypsiboas prasinus
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
W A N C Y S D
Barcode region
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)
AnW AnD
Nested or Semi-nested PCR- Museum specimens
COI
W A N C Y S D
Barcode region
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)
16S
LV16S
An-16SF An-16SR
COI
W A N C Y S D
Barcode region
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)
AnW AnD
COI and 16S - All species amplified
Primers design: Results
0
15
30
45
60
AnF1+AnCOIR AnF1+AnR1 AW-AD 16S
Works Fail
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)
Number species Anura:
58
Number individuals:
170
~3 ind/ sp
Intra and interspecific diversity – COI x 16S
16S
COI
Barcode region
COI: 0.00-0.135 (K2P) 16S: 0.00-0.18 (K2P)
Intraspecific diversity – COI x 16S
0
0,04
0,08
0,12
0,16
COI 16S
K2
P d
ista
nc
e
COI: 0.00-0.135 (K2P) 16S: 0.00-0.18 (K2P)
0
0,04
0,08
0,12
0,16
COI 16S
Intraspecific diversity – COI x 16SK
2P
dis
tan
ce
Highly correlated with geographical distance
0
0,04
0,08
0,12
0,16
COI 16S
Intraspecific diversity – COI x 16SK
2P
dis
tan
ce
Highly correlated with geographical distance
Potential new species? – look for other evidences
0
0,04
0,08
0,12
0,16
COI 16S
Intraspecific diversity – COI x 16SK
2P
dis
tan
ce
COI
Interspecific variation
15-29%
0
2
4
6
8
10
12
14
16
18
20
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
Intra Inter
0
2
4
6
8
10
12
14
16
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
intra inter
16S
Interspecific variation
6-30%
Nº
specie
sN
º specie
s
Interspecific diversity – COI x 16S
K2P distance
K2P distance
Candidate new species?
Sc. catharinae Pseudopadicola cf. falcipes
COI
Interspecific variation
15-29%
16S
Interspecific variation
6-30%
0
2
4
6
8
10
12
14
16
18
20
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
Intra Inter
0
2
4
6
8
10
12
14
16
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
intra inter
Nº
specie
sN
º specie
s
Interspecific diversity – COI x 16S
COI
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
Can not suggest a threshold
values to identify candidate
species based on this data
0
2
4
6
8
10
12
14
16
18
20
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
Intra Inter
0
2
4
6
8
10
12
14
16
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
intra inter
% variation
% variation
Nº
specie
sN
º specie
s
Interspecific diversity – COI x 16S
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)
21 specimens
http://www.iucnredlist.org/details/57151/0
Célio F B Haddad
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
A112 L.ocellatus_ES
A264 L.ocellatus_BA
A235 L.ocellatus_SC
A272 L.ocellatus_SP
A228 L.ocellatus_SP
A253 L.ocellatus_SP
A205 L.ocellatus_MG
A219 L.ocellatus_SP
A222 L.ocellatus_MG
A232 L.ocellatus_PR
A221 L.ocellatus_PR
A260 L.ocellatus_PR
A267 L.ocellatus_RS
A236 L.ocellatus_RS100
90
99
100
100
73
7898
100
96
0.02
Neighbor-joining/ K2P
AND
Gen. Mix.Yule-Coalescent model – GMYC
(Bayesian analysis)
Leptodactylus ocellatus: tree
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
A112 L.ocellatus_ES
A264 L.ocellatus_BA
A235 L.ocellatus_SC
A272 L.ocellatus_SP
A228 L.ocellatus_SP
A253 L.ocellatus_SP
A205 L.ocellatus_MG
A219 L.ocellatus_SP
A222 L.ocellatus_MG
A232 L.ocellatus_PR
A221 L.ocellatus_PR
A260 L.ocellatus_PR
A267 L.ocellatus_RS
A236 L.ocellatus_RS100
90
99
100
100
73
7898
100
96
0.02
COI – NJ K2P
Leptodactylus ocellatus: Clades
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
A112 L.ocellatus_ES
A264 L.ocellatus_BA
A235 L.ocellatus_SC
A272 L.ocellatus_SP
A228 L.ocellatus_SP
A253 L.ocellatus_SP
A205 L.ocellatus_MG
A219 L.ocellatus_SP
A222 L.ocellatus_MG
A232 L.ocellatus_PR
A221 L.ocellatus_PR
A260 L.ocellatus_PR
A267 L.ocellatus_RS
A236 L.ocellatus_RS100
90
99
100
100
73
7898
100
96
0.02
A181 16S BA
A037 16S 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 16S SP
A221 16S PR
A219 16S SP
A222 16S MG
A232 16S PR
A211 16S SC
A205 16S MG
A260 16S PR
A267 16S RS
A236 16S RS
100
89
68
88
99
0.02
COI – NJ K2P 16S – NJ K2P
Leptodactylus ocellatus: clades COI x 16S
clade 1
clade 2: D=0.016
clade 3: D=0.004
clade 4: D=0.006
clade 5: D=0.011
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
A112 L.ocellatus_ES
A264 L.ocellatus_BA
A235 L.ocellatus_SC
A272 L.ocellatus_SP
A228 L.ocellatus_SP
A253 L.ocellatus_SP
A205 L.ocellatus_MG
A219 L.ocellatus_SP
A222 L.ocellatus_MG
A232 L.ocellatus_PR
A221 L.ocellatus_PR
A260 L.ocellatus_PR
A267 L.ocellatus_RS
A236 L.ocellatus_RS100
90
99
100
100
73
7898
100
96
0.02
Leptodactylus ocellatus: intra-clade diversity
K2P
D=0.038
D=0.147
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
A112 L.ocellatus_ES
A264 L.ocellatus_BA
A235 L.ocellatus_SC
A272 L.ocellatus_SP
A228 L.ocellatus_SP
A253 L.ocellatus_SP
A205 L.ocellatus_MG
A219 L.ocellatus_SP
A222 L.ocellatus_MG
A232 L.ocellatus_PR
A221 L.ocellatus_PR
A260 L.ocellatus_PR
A267 L.ocellatus_RS
A236 L.ocellatus_RS100
90
99
100
100
73
7898
100
96
0.02
Leptodactylus ocellatus: inter-clade diversity
K2P
Strong correlated with geographical distribution
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
A112 L.ocellatus_ES
A264 L.ocellatus_BA
A235 L.ocellatus_SC
A272 L.ocellatus_SP
A228 L.ocellatus_SP
A253 L.ocellatus_SP
A205 L.ocellatus_MG
A219 L.ocellatus_SP
A222 L.ocellatus_MG
A232 L.ocellatus_PR
A221 L.ocellatus_PR
A260 L.ocellatus_PR
A267 L.ocellatus_RS
A236 L.ocellatus_RS100
90
99
100
100
73
7898
100
96
0.02
Leptodactylus ocellatus: geographic distribution
Bioacoustics and chromosomal differences
– coastal plain x plateau areas
(Silva et al. 2000)
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
A112 L.ocellatus_ES
A264 L.ocellatus_BA
A235 L.ocellatus_SC
A272 L.ocellatus_SP
A228 L.ocellatus_SP
A253 L.ocellatus_SP
A205 L.ocellatus_MG
A219 L.ocellatus_SP
A222 L.ocellatus_MG
A232 L.ocellatus_PR
A221 L.ocellatus_PR
A260 L.ocellatus_PR
A267 L.ocellatus_RS
A236 L.ocellatus_RS100
90
99
100
100
73
7898
100
96
0.02
Candidate new species?
Diversity is underestimated
Bioacoustics and chromosomal differences
– coastal plain x plateau areas
(Silva et al. 2000)
Candidate new species?
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
A112 L.ocellatus_ES
A264 L.ocellatus_BA
A235 L.ocellatus_SC
A272 L.ocellatus_SP
A228 L.ocellatus_SP
A253 L.ocellatus_SP
A205 L.ocellatus_MG
A219 L.ocellatus_SP
A222 L.ocellatus_MG
A232 L.ocellatus_PR
A221 L.ocellatus_PR
A260 L.ocellatus_PR
A267 L.ocellatus_RS
A236 L.ocellatus_RS100
90
99
100
100
73
7898
100
96
0.02
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
A258 S.fuscov SP
A200 S.fuscov SP
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
A247 S.fuscov MT
A226 S.fuscov MG
A266 S.fuscov RS
A241 S.fuscov SP
A197 S.fuscov SP
67
80
89
100
100
92
9691
96
63 49
47
38
54
15
11
34
33
38
2
1
3
6
13
0.02
COI: NJ-K2P
Scinax fuscovarius: tree
Diversity
0 – 24%
A258 S.fuscov SP
A200 S.fuscov SP
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
A247 S.fuscov MT
A226 S.fuscov MG
A266 S.fuscov RS
A241 S.fuscov SP
A197 S.fuscov SP
67
80
89
100
100
92
9691
96
63 49
47
38
54
15
11
34
33
38
2
1
3
6
13
0.02
COI: NJ-K2P
Scinax fuscovarius: geographic distribution
Not correlated with geographic distance
Specimens collection:
Correction
A197 Scinax sp.
A226 Sc. eurydice
A241 Sc. cf. hayii
A266 Scinax sp.
A258 S.fuscov SP
A200 S.fuscov SP
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
A247 S.fuscov MT
A226 S.fuscov MG
A266 S.fuscov RS
A241 S.fuscov SP
A197 S.fuscov SP
67
80
89
100
100
92
9691
96
63 49
47
38
54
15
11
34
33
38
2
1
3
6
13
0.02
COI: NJ-K2P
Scinax fuscovarius: collection
COI: NJ-K2P
Scinax fuscovarius: synonymy
A258 S.fuscov SP
A200 S.fuscov SP
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
A247 S.fuscov MT
89
100
100
92
9691
96
63 49
47
38
54
15
11
34
33
38
2
1
3
6
13
0.02
Diversity:
3.6 - 4.4%
COI: NJ-K2P
Scinax fuscovarius: synonymy
A258 S.fuscov SP
A200 S.fuscov SP
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
A247 S.fuscov MT
89
100
100
92
9691
96
63 49
47
38
54
15
11
34
33
38
2
1
3
6
13
0.02
Diversity:
3.6 - 4.4%
Scinax megapodia x Scinax fuscovarius
A247 - Sc. megapodia(?) distribution
Synonym revalidation?
Look for other evidences
COI: NJ-K2P
Scinax fuscovarius: synonymy
A258 S.fuscov SP
A200 S.fuscov SP
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
A247 S.fuscov MT
89
100
100
92
9691
96
63 49
47
38
54
15
11
34
33
38
2
1
3
6
13
0.02
Diversity:
3.6 - 4.4%
COI: NJ-K2P
Scinax fuscovarius: criptic diversity?
A258 S.fuscov SP
A200 S.fuscov SP
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 GO89
100
92
9691
96
63 49
47
38
54
15
11
34
33
38
2
1
3
6
13
0.02
Diversity
~2.6%
COI: NJ-K2P
Scinax fuscovarius: criptic diversity?
A258 S.fuscov SP
A200 S.fuscov SP
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 GO89
100
92
9691
96
63 49
47
38
54
15
11
34
33
38
2
1
3
6
13
0.02
COI: NJ-K2P
Scinax fuscovarius: criptic diversity?
A258 S.fuscov SP
A200 S.fuscov SP
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 GO89
100
92
9691
96
63 49
47
38
54
15
11
34
33
38
2
1
3
6
13
0.02
Geographic distribution of diversity?
Look for other evidences
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.
SUMARY AND CONCLUSIONS
Obrigada
Thank you
CNPq
Conselho Nacional de Desen volvimentoCi ent ífico e Te cno lógico
MLL Fellowship
Prodoc nº 563.975/05-9
Laboratory projects UNICAMP
Phylomedusa lemur –
Photo: ANAM
CFBH collection- UNESP
Barcoding Anurans from Brazilian Atlantic Forest
Mariana L. Lyra, Célio F. B. Haddad & Ana Maria L. de Azeredo-Espin
marilyra@unicamp.br
Capes/CNPq Prodoc nº 563.975/05-9
International Symposium on DNA Barcoding –São Paulo 03-04 Dezember 2009
Ultrametric tree - MrBayes 3.2
Analise no R (split, ape, gee)
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
“Single thresholds”: 6 unidades coalescentes
Outgroup
“Multiple thresholds”: 7 unidades coalescentes
Outgroup
A m a z o n i a
C e r r a d o
Coastal Ecosystems
Coastal Ecosystems
Southern Grasslands
Pantanal
A t
l a
n t i c
F
o r
e s
t
t r a
n s
i t i
o n
z o
n e s
Created a new account
Order: Anura
15 families
76 species
Order: Gymnophiona
1 family
3 species
Order: Caudata
1 family
1 species
Total: 80 sp.
“ DNA Barcoding Anuran from Brazilian of Atlantic forest” Fellowship: Capes/CNPqProdoc nº 563.975/05-9