DNA barcoding of South American mammals

The Biota-FAPESP International Symposium on DNA Barcoding 29 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. Gathering data on components of native biodiversity.. Baseline data.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:. 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: 25 Beginning of the Brazilian DNA barcoding network - Large-scale inventory of Brazilian biodiversity - Multi-center project designed in 25 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 (25) Sampling strategy 5 sites: -, 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 4 Centers of Molecular Biodiversity: - Network of genome centers and molecular biology laboratories performing high-throughput sequencing of DNA barcodes from 5, samples; Bioinformatics tools: online management of data collection, analysis and integration (e.g. morphology, DNA barcodes, geography) Training activities: courses, workshops.

The effort towards Large-scale DNA barcoding of Neotropical mammals History: Regional CBOL meeting in Campinas, Brazil - Mammals emerge as a CBOL-endorsed regional priority for DNA barcoding. - Several members of the 25 network confirm participation, while additional partner institutions join.

The effort towards Large-scale DNA barcoding of Neotropical mammals History: present Activities:. Talks, symposia and/or planning meetings during subsequent regional, national and international congresses: -, 28 Brazilian Congress of Genetics - 28 Brazilian Congress of Zoology, Brazilian Congress of Mammalogy, Regional Genetics Meeting (RS) - 29 International Mammalogical Congress, Argentina 2. Funding proposals: CYTED (), PROSUL (, 28).

PROSUL project proposal 28 (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: 2 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 28 participating institutions Brazil PUCRS - Pontifícia Universidade Católica do Rio Grande do Sul UFMG - Universidade Federal de Minas Gerais UFRGS - Universidade Federal do Rio Grande do Sul UFES - Universidade Federal do Espírito Santo FZB/RS - Fundação Zoobotânica do Rio Grande do Sul URI Universidade Regional Integrada FURB Universidade Regional de Blumenau UFPR Universidade Federal do Paraná UFRJ - Museu Nacional INCA - Instituto Nacional de Câncer FIOCRUZ - Fundação Oswaldo Cruz UERJ - Universidade do Estado do Rio de Janeiro USP - Instituto de Biociências USP/ESALQ - Escola Superior de Agricultura Luiz de Queiroz INPA - Instituto Nacional de Pesquisas da Amazônia UFMS - Universidade Federal de Mato Grosso do Sul UCB-DF - Universidade Católica de Brasília UFPA - Universidade Federal do Pará UFAM - Universidade Federal do Amazonas

PROSUL 28 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. 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. Goiás/Brasília - UFG Uruguay. Universidad de la Republica Rodentia Primates Primates Carnivora Xenarthra Rodentia Chiroptera Small mammals Xenarthra Chiroptera Xenarthra Chiroptera Rodentia

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 5 spp. Total: 9 spp (~ 6 specimens) Belo Horizonte (UFMG) Xenarthra spp. Chiroptera 35 spp. (> 2 specimens) Espírito Santo (UFES) Rodentia, Didelphimorphia, Chiroptera 85 spp. (259 specimens) São Paulo (USP) Xenarthra 6 spp. (49 specimens) [N. Moraes-Barros] Next targets: Chiroptera, Rodentia [F. Martins]

6 99 6 8 8 98 99 99 83 86 4 bpgy35 bpgy6 Pgy5 Pgy2 bpve5 Lycalopex gymnocercus bpve6 Lycalopex vetulus 99 bpve4 bcth28 98 bcth25 bcth64 Cerdocyon thous bcthimperatrizma bcth85 92 82 bcth5 Lobo-guaraExtrSCL bcbr32ii Chrysocyon brachyurus Lobo-guara3 Cbr5 Aau586 Aau683 Aau68 Arctocephalus australis 8 A.australis Aau689L-59-H4 Arctocephalus forsteri bpbr2 Pteronura brasiliensis bpbr Martes americana Martes americana (Neártico) Mam2 Lcn Lontra canadensis (Neártico) Lontra canadensis bllo3 bllo6 bllo5 Lontra longicaudis bllo29 bllo3 bpon56 Panthera onca bp3 Panthera tigris Puma concolor Lco9 Leopardus colocolo Lco Oge38 blge32 Leopardus geoffroyi Oge63 blti9 blge Leopardus tigrinus blti96 99 99 62 9 Building a carnivore barcode database: COI.2

8 32 39 34 86 46 52 98 58 85 blge28 Leopardus geoffroyi blge3 Leopardus geoffroyi blge29 Leopardus geoffroyi blge36 Leopardus geoffroyi blge43 Leopardus geoffroyi blg68 Leopardus geoffroyi blge65 Leopardus geoffroyi blge66 Leopardus geoffroyi blge5 Leopardus geoffroyi blge54 Leopardus geoffroyi blge55 Leopardus geoffroyi blti59 Leopardus tigrinus Brazil f Bolívia Urugua y L. g e o f r o y i 99 86 blti5 Leopardus tigrinus blti62 Leopardus tigrinus blti Leopardus tigrinus Lco Leopardus colocolo Lpa32 Leopardus pardalis Leopardus tigrinus Leopardus colocolo Leopardus pardalis.2

6 9 55 3 86 84 59 8 43 bpve35 Lycalopex vetulus Pve3 Lycalopex vetulus bpve3 Lycalopex vetulus Pve3 Lycalopex vetulus bpve35 Lycalopex vetulus bpve3 Lycalopex vetulus Pve35 Lycalopex vetulus culp28 Lycalopex culpaeus culp38 Lycalopex culpaeus 8 6 culp66 Lycalopex culpaeus culp285 Lycalopex culpaeus bpgy4 Lycalopex gymnocercus 98 36 52 6 63 89 3 bpgy9 Lycalopex gymnocercus s. Lycalopex sechurea s. Lycalopex sechurea s.2 Lycalopex sechurea Dg9 Lycalopex griseus Dg8coi Lycalopex griseus Dgr5 Lycalopex griseus Dgr9 Lycalopex griseus Dfu29 Lycalopex fulvipes Dfu29. Lycalopex fulvipes Df Lycalopex fulvipes 56 Dfu24 Lycalopex fulvipes Df2 Lycalopex fulvipes Dfu3 Lycalopex fulvipes 96 bpgy35 Lycalopex gymnocercus Dgr8 Lycalopex griseus Lycalopex vetulus Lycalopex culpaeus Lycalopex sechurae Lycalopex fulvipes Lycalopex gymnocercus Lycalopex griseus.2

Evolutionary ecology of L. tigrinus and L. geoffroyi? Leopardus tigrinus L. colocolo Leopardus geoffroyi

Habitat use of L. tigrinus and L. geoffroyi in RS state Argentina N Uruguay miles Atlantic Ocean Gramineous Steppe Park Steppe Arboreal Steppe Steppical Savannah Semidecidual Stational Forest Decidual Stational Forest Mixed Ombrophilous Forest Dense Ombrophilous Forest Pioneer Formations Ecological Tension Area

% of prey in diet Dietary analysis of L. tigrinus e L. geoffroyi using stomach contents of roadkilled animals 3 25 Leopardus tigrinus Leopardus geoffroyi 2 5 5 Akodon sp Calomys sp Delomys dorsalis Holochilus brasiliensis Mus musculus Oligorymozys sp Oryzomys sp Oxymycterus sp Rattus rattus Cavia sp Monodelphis sp Prey category Columbiformes Cuculiformes Gruiformes Passeriformes Tinamiformes Sauria Serpentes Anura

Dietary analysis of L. tigrinus e L. geoffroyi using stomach contents of roadkilled animals: DNA barcoding of prey items 42 3 2 2 6 93 8 99 99 99 8 96 39 CE Puma yagouaroundi Ne36 CE2 Puma yagouaroundi Ne36 OPN5 Oligoryzomys nigripes OPN8 Oligoryzomys nigripes OPN55 Oligoryzomys nigripes JR332 Oligoryzomys flavescens JR2 Oligoryzomys flavescens JR29 Oligoryzomys flavescens JR22 Oligoryzomys flavescens OPN6 Oligoryzomys nigripes JR244 Oligoryzomys flavescens CE32 L. geoffroyi Lgeof8 CE39 L. geoffroyi NE32 CE4 L. tigrinus blti22 EU95456 Oligoryzomys fulvescens PCE2 Deltamys kempi CE L. tigrinus NE32 CE2 L. tigrinus blti22 CE3 Leopardus geoffroyi Lge6 CE4 Leopardus geoffroyi Lge6 JR96 Sooretamys angouya 99 Nectomys squamipes CE6 L. tigrinus blti 89 68 45 99 8 86 8 65 92 59 99 58 CE63 L. tigrinus NE22 935 Juliomys pictipes 966 Juliomys pictipes 56 8 99 43 5 99 96 55 82 85 68 CE25 L. tigrinus blti9 CE65 L. tigrinus NE22 CE5 L. geoffroyi blge4 CE59 L. tigrinus blti69 JR 45 Calomys tener LBCE 5556 Calomys expulsus CRB 32 Calomys expulsus LBCE 54 Calomys expulsus CE Leopardus geoffroyi Ne32 LBCE 548 Calomys expulsus CE66 L. tigrinus NE33 CE62 L. tigrinus NE22 JR395 Oxymycterus nasutus JR328 Akodon azarae JR329 Akodon azarae 9595 Akodon montensis PCE23 Oligoryzomys nigripes JR 4 Akodon montensis CE6 Leopardus geoffroyi Ne38 6844 Akodon paranaensis 6963 Akodon paranaensis CE9 P. yagouaroundi NE34 JR4 Akodon paranaensis.2

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 bpon56 (fezes Panthera onca em álcool) bpon55 (fezes de Panthera onca em DET) bpon36 (fezes de Panthera onca) bpon3 (fezes de Panthera onca) bpon56 (fezes Panthera onca - 98 mg) bpon32 (sangue de Panthera onca) bpon56 (fezes de Panthera onca em DET) P3-6 (pele de Panthera onca) bpon35 (fezes de Panthera onca) P3-2 (pêlos de Panthera onca) bpon56 (fezes de Panthera onca - 48 mg) bpon24 (sangue de Panthera onca) P3- (pêlos de Panthera onca) F2-636 (fezes de felino grande) 99 Puma concolor (sangue) L.V.(fezes de felino grande) F5-C (fezes de felino grande) F2-635 (fezes de felino grande) Felis catus.2 Haag et al. 29

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.

Identification of carnivores using short DNA segments Ongoing surveys and ecological studies

Identification of carnivores using short DNA segments Testing and optimization of protocols for African carnivores: Partnership with CCF, Namibia.

25 carr Reverse Complement 2 Car8 2 bfca5 3 bfca53 34 5 bfca52 bfca54 46 Car3 bfca59 8 6 9 9 46 52 Lse bfca55 bfca58 bfca2 bfca56 NC. Felis catus Fma Fma 6Lse8 99 9 Fni serval 3 serval Fni4 AY5984. AY5984S Lynx canadensis AH4. SEG AY5984S Lynx canadensis Car69 Cca2 Cca CarCaracal Car49 car88r Reverse Complement 5 33 4 23 39 car93r Reverse Complement car54copy car4 car6r Reverse Complement Aju NC 522. Acinonyx jubatus Car48 AY463959. Acinonyx jubatus car8r Reverse Complement AF34483. Acinonyx jubatus car89r Reverse Complement car94r Reverse Complement car2r Reverse Complement Car2 88 52 Ppa286 25 carr Reverse Complement 2 Car8 2 bfca5 3 bfca53 bfca52 34 5 bfca54 46 Car3 bfca59 3 2 5 2 42 9 6 39 9 38 25 48 5 35 Car8ccfAndBrazil CarLeop 88 Rme Ple85 Ple53 9 5 Hja 88 car84copy Reverse Complement lionf car52 Phe Hpa Pcr Hbr Cfe NC 845. Neofelis nebulosa Hhy4 Ccr34 Ccr236 Pli Vci babi Ial2 NC 62. Halichoerus grypus Zca Oro AY329. Hydrurga leptonyx AY3296. Hydrurga leptonyx NC 8425. Hydrurga leptonyx 8 9 bfca55 52 bfca58 39 bfca2 bfca56 NC. Felis catus Fma 45 6 5 95 Man68 AY3295. Ommatophoca rossii AY3294. Ommatophoca rossii Bas Bal 9 Fma AM898. Spilogale putorius complete bcar24 29 AB295. Meles meles anakuma AB296. Martes melampus AB29. Enhydra lutris Ist Ssu Car2 8 23 AY59856. AY59856S Alopex lagopus AH43. SEG AY59856S Alopex lagopus CarBatEaredFox 45 Band2and5 carwilddog DQ4858. Canis lupus isolate fro... 6 AY598494. AY598494S Canis lupus DQ4852. Canis familiaris isolate... AH42. SEG AY598494S Canis lupus 38 35 43 DQ485. Canis lupus isolate 2 fro... DQ4854. Canis lupus isolate fro... DQ485. Canis familiaris isolate... AY65638. Canis familiaris isolate... 28 AY2988. Canis familiaris AY6563. Canis familiaris isolate... NC 28.4 Canis lupus familiaris DQ4849. Canis familiaris isolate... DQ4855. Canis lupus isolate fro... 58 DQ48492. Canis familiaris isolate... DQ4853. Canis lupus isolate fro... NC 892. Canis lupus 86 DQ4856. Canis lupus isolate fro... car5copy 85 8 car85r Reverse Complement AY65639. Canis familiaris isolate... NC 893. Canis latrans DQ485. Canis latrans isolate 2 f... 99 DQ485. Canis latrans isolate f... DQ4859. Canis latrans isolate f... Car9 45 car23 copy 8 Car53 Car8 2 car4r Reverse Complement 3 Car car63r Reverse Complement Car5 Car56 Car car9r Reverse Complement 2 Car82 car45r Reverse Complement Car9ccf Car64 Car68 2 Car5 Car8 car9r Reverse Complement Car43 Car83 Car car44copy car55copy 48 car62 car66 car65copy 6 car6copy 46 34 Car5 IUCN Cat Specialist Group

8 9 9 46 46 25 carr Reverse Complement 2 Car8 2 bfca5 3 34 52 9 39 Lse bfca53 bfca52 bfca54 Car3 bfca59 bfca55 bfca58 bfca2 bfca56 NC. Felis catus Fma Fma 6Lse8 5 Fni serval 3 serval Fni4 AY5984. AY5984S Lynx canadensis AH4. SEG AY5984S Lynx canadensis Car69 88 52 Cca2 Cca CarCaracal Car49 car88r Reverse Complement 5 33 car93r Reverse Complement car54copy car4 car6r Reverse Complement 4 Aju NC 522. Acinonyx jubatus 23 Car48 AY463959. Acinonyx jubatus car8r Reverse Complement AF34483. Acinonyx jubatus car89r Reverse Complement car94r Reverse Complement 99 car2r Reverse Complement 6 Car2 Ppa286 5 Car8ccfAndBrazil 9 35 CarLeop Ple85 48 Ple53 car84copy Reverse Complement 88 5 lionf Cfe car52 3 NC 845. Neofelis nebulosa 5 Vci 38 babi 9 Ial2 Rme 9 Hja 2 Hpa 2 Pcr Hbr 39 Hhy4 Ccr34 25 88 Ccr236 Phe Pli 6 NC 62. Halichoerus grypus 42 Zca Oro AY329. Hydrurga leptonyx AY3296. Hydrurga leptonyx NC 8425. Hydrurga leptonyx 45 Man68 AY3295. Ommatophoca rossii 5 AY3294. Ommatophoca rossii 95 Bal 6 Bas AM898. Spilogale putorius complete bcar24 AB296. Martes melampus 29 AB295. Meles meles anakuma AB29. Enhydra lutris Ist Ssu Car2 8 AY59856. AY59856S Alopex lagopus AH43. SEG AY59856S Alopex lagopus 23 CarBatEaredFox carwilddog 45 Band2and5 DQ4858. Canis lupus isolate fro... 6 AY598494. AY598494S Canis lupus DQ4852. Canis familiaris isolate... AH42. SEG AY598494S Canis lupus Car69 Cca2 Cca 88 52 Caracal 38 35 43 DQ485. Canis lupus isolate 2 fro... DQ4854. Canis lupus isolate fro... DQ485. Canis familiaris isolate... AY65638. Canis familiaris isolate... 28 AY2988. Canis familiaris AY6563. Canis familiaris isolate... NC 28.4 Canis lupus familiaris DQ4849. Canis familiaris isolate... DQ4855. Canis lupus isolate fro... 58 DQ48492. Canis familiaris isolate... DQ4853. Canis lupus isolate fro... NC 892. Canis lupus 86 DQ4856. Canis lupus isolate fro... car5copy 85 8 car85r Reverse Complement AY65639. Canis familiaris isolate... NC 893. Canis latrans DQ485. Canis latrans isolate 2 f... 99 DQ485. Canis latrans isolate f... DQ4859. Canis latrans isolate f... Car9 45 car23 copy 8 Car53 Car8 2 car4r Reverse Complement 3 Car car63r Reverse Complement Car5 Car56 Car car9r Reverse Complement 2 Car82 car45r Reverse Complement Car9ccf Car64 Car68 2 Car5 Car8 car9r Reverse Complement Car43 Car83 Car car44copy car55copy 48 car62 car66 car65copy 6 car6copy 46 34 Car5

carr Reverse Complement 8 9 9 46 46 25 2 Car8 2 bfca5 3 34 52 9 39 Lse bfca53 bfca52 bfca54 Car3 bfca59 bfca55 bfca58 bfca2 bfca56 NC. Felis catus Fma Fma 6Lse8 5 Fni serval 3 serval Fni4 AY5984. AY5984S Lynx canadensis AH4. SEG AY5984S Lynx canadensis Car69 88 52 Cca2 Cca CarCaracal Car49 car88r Reverse Complement 5 33 car93r Reverse Complement car54copy car4 car6r Reverse Complement 4 Aju NC 522. Acinonyx jubatus 23 Car48 AY463959. Acinonyx jubatus car8r Reverse Complement AF34483. Acinonyx jubatus car89r Reverse Complement car94r Reverse Complement 99 car2r Reverse Complement 6 Car2 Ppa286 5 Car8ccfAndBrazil 9 35 CarLeop Ple85 48 Ple53 car84copy Reverse Complement 88 5 lionf Cfe car52 3 NC 845. Neofelis nebulosa 5 Vci 38 babi 9 Ial2 Rme 9 Hja 2 Hpa 2 Pcr Hbr 39 Hhy4 Ccr34 25 88 Ccr236 Phe Pli 6 NC 62. Halichoerus grypus 42 Zca Oro AY329. Hydrurga leptonyx AY3296. Hydrurga leptonyx NC 8425. Hydrurga leptonyx 45 Man68 AY3295. Ommatophoca rossii 5 AY3294. Ommatophoca rossii 95 Bal 6 Bas AM898. Spilogale putorius complete bcar24 AB296. Martes melampus 29 AB295. Meles meles anakuma AB29. Enhydra lutris Ist Ssu Car2 8 AY59856. AY59856S Alopex lagopus AH43. SEG AY59856S Alopex lagopus 23 CarBatEaredFox carwilddog 45 Band2and5 DQ4858. Canis lupus isolate fro... 6 AY598494. AY598494S Canis lupus DQ4852. Canis familiaris isolate... AH42. SEG AY598494S Canis lupus DQ485. Canis lupus isolate 2 fro... 38 DQ4854. Canis lupus isolate fro... DQ485. Canis familiaris isolate... AY65638. Canis familiaris isolate... 28 AY2988. Canis familiaris AY6563. Canis familiaris isolate... NC 28.4 Canis lupus familiaris 35 DQ4849. Canis familiaris isolate... DQ4855. Canis lupus isolate fro... 58 DQ48492. Canis familiaris isolate... DQ4853. Canis lupus isolate fro... NC 892. Canis lupus 86 DQ4856. Canis lupus isolate fro... car5copy 85 8 car85r Reverse Complement AY65639. Canis familiaris isolate... NC 893. Canis latrans DQ485. Canis latrans isolate 2 f... 99 DQ485. Canis latrans isolate f... 43 DQ4859. Canis latrans isolate f... Car9 45 car23 copy 8 Car53 Car8 2 car4r Reverse Complement 3 Car Car49 car88r Reverse Complement 9 car93r Reverse Complement 46 car54copy 5 car4 33 car6r Reverse Complement 4 Aju NC 522. Acinonyx jubatus 23 Car48 AY463959. Acinonyx jubatus car8r Reverse Complement AF34483. Acinonyx jubatus car89r Reverse Complement car94r Reverse Complement car63r Reverse Complement Car5 Car56 Car car9r Reverse Complement 2 Car82 car45r Reverse Complement Car9ccf Car64 Car68 2 Car5 Car8 car9r Reverse Complement Car43 Car83 Car car44copy car55copy 48 car62 car66 car65copy 6 car6copy 46 34 Car5

carr Reverse Complement 8 9 9 46 46 25 2 Car8 2 bfca5 3 34 52 9 39 Lse bfca53 bfca52 bfca54 Car3 bfca59 bfca55 bfca58 bfca2 bfca56 NC. Felis catus Fma Fma 6Lse8 5 Fni serval 3 serval Fni4 AY5984. AY5984S Lynx canadensis AH4. SEG AY5984S Lynx canadensis Car69 88 52 Cca2 Cca CarCaracal Car49 car88r Reverse Complement 5 33 car93r Reverse Complement car54copy car4 car6r Reverse Complement 4 Aju NC 522. Acinonyx jubatus 23 Car48 AY463959. Acinonyx jubatus car8r Reverse Complement AF34483. Acinonyx jubatus car89r Reverse Complement car94r Reverse Complement 99 car2r Reverse Complement 6 Car2 Ppa286 5 Car8ccfAndBrazil 9 35 CarLeop Ple85 48 Ple53 car84copy Reverse Complement 88 5 lionf Cfe car52 3 NC 845. Neofelis nebulosa 5 Vci 38 babi 9 Ial2 Rme 9 Hja 2 Hpa 2 Pcr Hbr 39 Hhy4 Ccr34 25 88 Ccr236 Phe Pli 6 NC 62. Halichoerus grypus 42 Zca Oro AY329. Hydrurga leptonyx AY3296. Hydrurga leptonyx NC 8425. Hydrurga leptonyx 45 Man68 AY3295. Ommatophoca rossii 5 AY3294. Ommatophoca rossii 95 Bal 6 Bas AM898. Spilogale putorius complete bcar24 AB296. Martes melampus 29 AB295. Meles meles anakuma AB29. Enhydra lutris Ist Ssu Car2 8 AY59856. AY59856S Alopex lagopus AH43. SEG AY59856S Alopex lagopus 23 CarBatEaredFox carwilddog 45 Band2and5 DQ4858. Canis lupus isolate fro... 6 AY598494. AY598494S Canis lupus DQ4852. Canis familiaris isolate... AH42. SEG AY598494S Canis lupus DQ485. Canis lupus isolate 2 fro... 38 DQ4854. Canis lupus isolate fro... DQ485. Canis familiaris isolate... AY65638. Canis familiaris isolate... 28 AY2988. Canis familiaris AY6563. Canis familiaris isolate... NC 28.4 Canis lupus familiaris 35 DQ4849. Canis familiaris isolate... DQ4855. Canis lupus isolate fro... 58 DQ48492. Canis familiaris isolate... DQ4853. Canis lupus isolate fro... NC 892. Canis lupus 86 DQ4856. Canis lupus isolate fro... car5copy 85 8 car85r Reverse Complement AY65639. Canis familiaris isolate... NC 893. Canis latrans DQ485. Canis latrans isolate 2 f... 99 DQ485. Canis latrans isolate f... 43 DQ4859. Canis latrans isolate f... Car9 45 car23 copy 8 Car53 Car8 2 car4r Reverse Complement 3 Car car63r Reverse Complement Car5 Car56 Car car9r Reverse Complement 2 Car82 car45r Reverse Complement Car9ccf Car64 Car68 2 Car5 Car8 car9r Reverse Complement Car43 Car83 Car car44copy car55copy 48 car62 car66 car65copy 6 car6copy 46 34 Car5 9 99 car2r Reverse Complement 6 Car2 Ppa286 5 35 Car8 CarLeop Ple85 48 Ple53 88 car84 5 lionf

carr Reverse Complement 8 3 2 23 5 2 42 45 6 29 8 9 6 5 39 45 9 38 25 2 Car8 2 bfca5 Lse bfca53 bfca52 bfca54 Car3 bfca59 bfca55 bfca58 bfca2 bfca56 NC. Felis catus Fma Fma 6Lse8 Fni serval 3 serval Fni4 AY5984. AY5984S Lynx canadensis AH4. SEG AY5984S Lynx canadensis Car69 Cca2 Cca CarCaracal Car49 car88r Reverse Complement car93r Reverse Complement car54copy car4 car6r Reverse Complement Aju NC 522. Acinonyx jubatus Car48 AY463959. Acinonyx jubatus car8r Reverse Complement AF34483. Acinonyx jubatus car89r Reverse Complement car94r Reverse Complement car2r Reverse Complement Car2 Ppa286 Car8ccfAndBrazil CarLeop Rme Ple85 Ple53 Hja car84copy Reverse Complement lionf car52 Phe Hpa Pcr Hbr Cfe NC 845. Neofelis nebulosa Hhy4 Ccr34 Ccr236 Pli Vci babi Ial2 NC 62. Halichoerus grypus Man68 Zca Oro AY329. Hydrurga leptonyx AY3296. Hydrurga leptonyx NC 8425. Hydrurga leptonyx AY3295. Ommatophoca rossii AY3294. Ommatophoca rossii Bas Bal AM898. Spilogale putorius complete bcar24 AB295. Meles meles anakuma 6 25 6 48 9 9 5 35 46 88 38 9 95 46 5 3 34 52 99 9 5 33 4 23 39 88 35 5 Ist 43 88 52 AB296. Martes melampus AB29. Enhydra lutris Ssu Car2 8 2 AY59856. AY59856S Alopex lagopus AH43. SEG AY59856S Alopex lagopus CarBatEaredFox Band2and5 8 3 45 99 Car9 86 Car53 carwilddog DQ4858. Canis lupus isolate fro... AY598494. AY598494S Canis lupus DQ4852. Canis familiaris isolate... AH42. SEG AY598494S Canis lupus DQ485. Canis lupus isolate 2 fro... DQ4854. Canis lupus isolate fro... DQ485. Canis familiaris isolate... AY65638. Canis familiaris isolate... AY2988. Canis familiaris AY6563. Canis familiaris isolate... NC 28.4 Canis lupus familiaris DQ4849. Canis familiaris isolate... DQ4855. Canis lupus isolate fro... DQ48492. Canis familiaris isolate... DQ4853. Canis lupus isolate fro... NC 892. Canis lupus DQ4856. Canis lupus isolate fro... car5copy 85 car85r Reverse Complement AY65639. Canis familiaris isolate... NC 893. Canis latrans DQ485. Canis latrans isolate 2 f... DQ485. Canis latrans isolate f... DQ4859. Canis latrans isolate f... Car8 car23 copy car4r Reverse Complement Car car63r Reverse Complement Car5 Car Car56 car9r Reverse Complement 2 Car82 car45r Reverse Complement Car64 Car68 2 Car5 48 6 Car8 Car9ccf car9r Reverse Complement Car43 Car83 Car car44copy car55copy car62 car66 46 34 58 28 car65copy car6copy Car5 43 DQ4849. Canis familiaris isolate... DQ4855. Canis lupus isolate fro... 58 DQ48492. Canis familiaris isolate... DQ4853. Canis lupus isolate fro... NC 892. Canis lupus 86 DQ4856. Canis lupus isolate fro... car5 8 85car85R AY65639. Canis familiaris isolate... NC 893. Canis latrans DQ485. Canis latrans isolate 2 f... 99 DQ485. Canis latrans isolate f... DQ4859. Canis latrans isolate f... Car9 car23 8 Car53 Car8 car4r Car car63r Car5 Car56 Car car9r Car82 car45r Car9ccf Car64 Car68 Car5 Car8 car9r Car43 Car83 Car car44copy car55copy car62 car66 car65copy car6copy Car5

Current challenges for large-scale DNA barcoding of Neotropical mammals:. 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.

ACKNOWLEDGEMENTS: Students Henrique Figueiró Marina Favarini Fernanda Valdez Ezequiel Fabiano Flávia Tirelli Cristine Trinca Camila Jaeger Paulo B. Chaves Vanessa G. Graeff Gabriel Macedo Carnivore scat ID projects Fernanda Michalski (Pró-Carnívoros) Laurie Marker (CCF) Anne Schmidt-Küntzel (CCF) Support: CNPq, CAPES, PUCRS

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)