The Use of Molecular Tools in Ecological Studies

International Workshop on Applied Ecology and Human Dimensions in Biological Conservation Biota FAPESP - 2009 The Use of Molecular Tools in Ecological Studies Eduardo Eizirik Faculdade de Biociências, PUCRS, Brasil Instituto Pró-Carnívoros, Brasil Laboratory of Genomic Diversity, NIH, USA

Molecular Markers in Conservation Biology. Inventories of biodiversity - Systematics, species delimitation - Species identification - DNA barcoding 2. Geographic distributions, community composition, habitat association 3. Monitoring of species and populations 4. Phylogeography, population structure and demographic history - Definition of units for conservation 5. Behavioral ecology (social structure, dispersal patterns) 6. Forensics 7. Adaptation and natural selection (including at the genomic level)

Different Markers for Different Tasks. Deep phylogenetics 2. Shallow phylogenetics and phylogeography 3. Species identification 4. Population genetics 5. Parentage/kinship analyses 6. Individual identification 7. Sexing 8. Pathogen identification 9. Analysis of genes involved in immune response 0. Analysis of genes involved in potentially adaptive phenotypes

Development of molecular tools for carnivore species identification from faecal DNA

Development of molecular tools for carnivore species identification from faecal DNA bpon56 (fezes Panthera onca em álcool) bpon55 (fezes de Panthera onca em DET) bpon306 (fezes de Panthera onca) bpon307 (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) bpon305 (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) F57-C (fezes de felino grande) F2-635 (fezes de felino grande) Felis catus 0.02 Haag et al. 2009

Identification of carnivores using short DNA segments Goal: global standardized saystem for carnivore identification. Application in ecological, biogeographic and forensic studies. Ongoing tests in multiple field sites in South America Brazil (RS, ES, MS, MT, TO, MG,DF), Argentina. P.B. Chaves et al., in prep.

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 car70r Reverse Complement 2 Car78 2 bfca57 30 bfca53 34 57 bfca52 bfca54 46 Car73 bfca59 0 0 8 7 7 6 9 97 46 70 52 Lse bfca55 bfca58 bfca02 bfca56 NC 00700. Felis catus Fma Fma 67Lse8 99 97 70 Fni serval 3 serval Fni4 AY598470. AY598470S0 Lynx canadensis AH04070. SEG AY598470S Lynx canadensis Car69 Cca2 Cca CarCaracal Car49 car88r Reverse Complement 50 33 4 23 39 car93r Reverse Complement car54copy car47 car76r Reverse Complement Aju NC 00522. Acinonyx jubatus Car48 AY463959. Acinonyx jubatus car87r Reverse Complement 0 AF344830. Acinonyx jubatus car89r Reverse Complement 7 car94r Reverse Complement car2r Reverse Complement Car2 88 52 Ppa286 25 car70r Reverse Complement 2 Car78 2 bfca57 30 bfca53 bfca52 34 57 bfca54 46 Car73 bfca59 0 27 3 2 5 2 42 7 9 6 39 9 38 27 25 48 57 35 Car8ccfAndBrazil CarLeop 88 Rme Ple85 Ple53 97 75 Hja 88 car84copy Reverse Complement lionf car52 Phe Hpa Pcr Hbr Cfe NC 008450. Neofelis nebulosa Hhy40 Ccr34 Ccr236 Pli Vci babi Ial2 NC 00602. Halichoerus grypus Zca Oro AY377297. Hydrurga leptonyx AY377296. Hydrurga leptonyx NC 008425. Hydrurga leptonyx 8 9 bfca55 70 52 bfca58 39 bfca02 bfca56 NC 00700. Felis catus Fma 45 6 50 95 Man68 AY377295. Ommatophoca rossii AY377294. Ommatophoca rossii Bas Bal 97 Fma 7 AM7898. Spilogale putorius complete 0 bcar24 29 AB29075. Meles meles anakuma AB29076. Martes melampus AB29077. Enhydra lutris 0 Ist Ssu 0 Car2 8 23 AY598506. AY598506S0 Alopex lagopus AH04073. SEG AY598506S Alopex lagopus CarBatEaredFox 45 Band2and5 carwilddog DQ480508. Canis lupus isolate fro... 6 AY598494. AY598494S0 Canis lupus DQ480502. Canis familiaris isolate... AH04072. SEG AY598494S Canis lupus 38 DQ480507. Canis lupus isolate 2 fro... DQ480504. Canis lupus isolate fro... DQ48050. Canis familiaris isolate... AY656738. Canis familiaris isolate... 28 AY729880. Canis familiaris AY656737. Canis familiaris isolate... NC 002008.4 Canis lupus familiaris 35 43 87 DQ48049. Canis familiaris isolate... DQ480505. Canis lupus isolate fro... 58 DQ480492. Canis familiaris isolate... DQ480503. Canis lupus isolate fro... NC 008092. Canis lupus 86 DQ480506. Canis lupus isolate fro... car50copy 85 car85r Reverse Complement AY656739. Canis familiaris isolate... NC 008093. Canis latrans DQ4805. Canis latrans isolate 2 f... 99 DQ48050. Canis latrans isolate f... DQ480509. Canis latrans isolate f... Car79 45 car23 copy 78 Car53 Car8 2 car74r Reverse Complement 30 Car7 car63r Reverse Complement 7 Car57 Car56 27 Car car9r Reverse Complement 2 Car82 car45r Reverse Complement Car9ccf Car64 Car68 2 Car5 Car80 car90r Reverse Complement Car43 Car83 Car77 car44copy car55copy 48 car62 7 car66 27 car65copy 6 car60copy 46 34 Car75 IUCN Cat Specialist Group

0 0 0 0 0 27 8 3 0 2 7 23 5 2 42 45 6 29 7 8 7 7 9 6 50 39 45 9 38 27 25 6 48 9 97 57 35 46 25 car70r Reverse Complement 2 Car78 2 bfca57 Lse bfca53 bfca52 bfca54 Car73 bfca59 bfca55 bfca58 bfca02 bfca56 NC 00700. Felis catus Fma Fma 67Lse8 Fni serval 3 serval Fni4 AY598470. AY598470S0 Lynx canadensis AH04070. SEG AY598470S Lynx canadensis Car69 Cca2 Cca CarCaracal Car49 car88r Reverse Complement car93r Reverse Complement car54copy car47 car76r Reverse Complement Aju NC 00522. Acinonyx jubatus Car48 AY463959. Acinonyx jubatus car87r Reverse Complement 0 AF344830. Acinonyx jubatus car89r Reverse Complement 7 car94r Reverse Complement car2r Reverse Complement Car2 Ppa286 Car8ccfAndBrazil CarLeop 88 Rme 95 46 Ple85 Ple53 97 70 75 30 34 70 52 99 97 50 33 4 23 39 Hja 88 57 car84copy Reverse Complement lionf car52 Phe Hpa Pcr Hbr Cfe NC 008450. Neofelis nebulosa Hhy40 Ccr34 Ccr236 Pli Vci babi Ial2 NC 00602. Halichoerus grypus Man68 Zca Oro AY377297. Hydrurga leptonyx AY377296. Hydrurga leptonyx NC 008425. Hydrurga leptonyx AY377295. Ommatophoca rossii AY377294. Ommatophoca rossii Bas Bal AM7898. Spilogale putorius complete bcar24 AB29075. Meles meles anakuma 6 Ist 88 52 AB29076. Martes melampus AB29077. Enhydra lutris Ssu Car2 AY598506. AY598506S0 Alopex lagopus AH04073. SEG AY598506S Alopex lagopus CarBatEaredFox Band2and5 carwilddog DQ480508. Canis lupus isolate fro... AY598494. AY598494S0 Canis lupus DQ480502. Canis familiaris isolate... AH04072. SEG AY598494S Canis lupus Car69 Cca2 Cca 88 52 Caracal 38 DQ480507. Canis lupus isolate 2 fro... DQ480504. Canis lupus isolate fro... DQ48050. Canis familiaris isolate... AY656738. Canis familiaris isolate... 28 AY729880. Canis familiaris AY656737. Canis familiaris isolate... NC 002008.4 Canis lupus familiaris 35 43 87 DQ48049. Canis familiaris isolate... DQ480505. Canis lupus isolate fro... 58 DQ480492. Canis familiaris isolate... DQ480503. Canis lupus isolate fro... NC 008092. Canis lupus 86 DQ480506. Canis lupus isolate fro... car50copy 85 car85r Reverse Complement AY656739. Canis familiaris isolate... NC 008093. Canis latrans DQ4805. Canis latrans isolate 2 f... 99 DQ48050. Canis latrans isolate f... DQ480509. Canis latrans isolate f... Car79 45 car23 copy 78 Car53 Car8 2 car74r Reverse Complement 30 Car7 car63r Reverse Complement 7 Car57 Car56 27 Car car9r Reverse Complement 2 Car82 car45r Reverse Complement Car9ccf Car64 Car68 2 Car5 Car80 car90r Reverse Complement Car43 Car83 Car77 car44copy car55copy 48 car62 7 car66 27 car65copy 6 car60copy 46 34 Car75

car70r Reverse Complement 0 0 0 0 0 27 8 3 0 2 7 23 5 2 42 45 6 29 7 8 7 7 9 6 50 39 45 9 38 27 25 6 48 9 97 57 35 46 25 2 Car78 2 bfca57 Lse bfca53 bfca52 bfca54 Car73 bfca59 bfca55 bfca58 bfca02 bfca56 NC 00700. Felis catus Fma Fma 67Lse8 Fni serval 3 serval Fni4 AY598470. AY598470S0 Lynx canadensis AH04070. SEG AY598470S Lynx canadensis Car69 Cca2 Cca CarCaracal Car49 car88r Reverse Complement car93r Reverse Complement car54copy car47 car76r Reverse Complement Aju NC 00522. Acinonyx jubatus Car48 AY463959. Acinonyx jubatus car87r Reverse Complement 0 AF344830. Acinonyx jubatus car89r Reverse Complement 7 car94r Reverse Complement car2r Reverse Complement Car2 Ppa286 Car8ccfAndBrazil CarLeop 88 Rme 95 46 Ple85 Ple53 97 70 75 30 34 70 52 99 97 50 33 4 23 39 Hja 88 57 car84copy Reverse Complement lionf car52 Phe Hpa Pcr Hbr Cfe NC 008450. Neofelis nebulosa Hhy40 Ccr34 Ccr236 Pli Vci babi Ial2 NC 00602. Halichoerus grypus Man68 Zca Oro AY377297. Hydrurga leptonyx AY377296. Hydrurga leptonyx NC 008425. Hydrurga leptonyx AY377295. Ommatophoca rossii AY377294. Ommatophoca rossii Bas Bal AM7898. Spilogale putorius complete bcar24 AB29075. Meles meles anakuma 6 38 35 Ist 88 52 AB29076. Martes melampus AB29077. Enhydra lutris Ssu Car2 AY598506. AY598506S0 Alopex lagopus AH04073. SEG AY598506S Alopex lagopus CarBatEaredFox Band2and5 58 28 carwilddog DQ480508. Canis lupus isolate fro... AY598494. AY598494S0 Canis lupus DQ480502. Canis familiaris isolate... AH04072. SEG AY598494S Canis lupus DQ480507. Canis lupus isolate 2 fro... DQ480504. Canis lupus isolate fro... DQ48050. Canis familiaris isolate... AY656738. Canis familiaris isolate... AY729880. Canis familiaris AY656737. Canis familiaris isolate... NC 002008.4 Canis lupus familiaris DQ48049. Canis familiaris isolate... DQ480505. Canis lupus isolate fro... DQ480492. Canis familiaris isolate... DQ480503. Canis lupus isolate fro... Car49 car88r Reverse Complement 97 car93r Reverse Complement 46 car54copy 50 car47 33 car76r Reverse Complement 4 Aju NC 00522. Acinonyx jubatus 23 Car48 AY463959. Acinonyx jubatus car87r Reverse Complement 0 AF344830. Acinonyx jubatus car89r Reverse Complement 7 car94r Reverse Complement 43 87 NC 008092. Canis lupus 86 DQ480506. Canis lupus isolate fro... car50copy 85 car85r Reverse Complement AY656739. Canis familiaris isolate... NC 008093. Canis latrans DQ4805. Canis latrans isolate 2 f... 99 DQ48050. Canis latrans isolate f... DQ480509. Canis latrans isolate f... Car79 45 car23 copy 78 Car53 Car8 2 car74r Reverse Complement 30 Car7 car63r Reverse Complement 7 Car57 Car56 27 Car car9r Reverse Complement 2 Car82 car45r Reverse Complement Car9ccf Car64 Car68 2 Car5 Car80 car90r Reverse Complement Car43 Car83 Car77 car44copy car55copy 48 car62 7 car66 27 car65copy 6 car60copy 46 34 Car75

car70r Reverse Complement 0 0 0 0 0 27 8 3 0 2 7 23 5 2 42 45 6 29 7 8 7 7 9 6 50 39 45 9 38 27 25 6 48 9 97 57 35 46 25 2 Car78 2 bfca57 Lse bfca53 bfca52 bfca54 Car73 bfca59 bfca55 bfca58 bfca02 bfca56 NC 00700. Felis catus Fma Fma 67Lse8 Fni serval 3 serval Fni4 AY598470. AY598470S0 Lynx canadensis AH04070. SEG AY598470S Lynx canadensis Car69 Cca2 Cca CarCaracal Car49 car88r Reverse Complement car93r Reverse Complement car54copy car47 car76r Reverse Complement Aju NC 00522. Acinonyx jubatus Car48 AY463959. Acinonyx jubatus car87r Reverse Complement 0 AF344830. Acinonyx jubatus car89r Reverse Complement 7 car94r Reverse Complement car2r Reverse Complement Car2 Ppa286 Car8ccfAndBrazil CarLeop 88 Rme 95 46 Ple85 Ple53 97 70 75 30 34 70 52 99 97 50 33 4 23 39 Hja 88 57 car84copy Reverse Complement lionf car52 Phe Hpa Pcr Hbr Cfe NC 008450. Neofelis nebulosa Hhy40 Ccr34 Ccr236 Pli Vci babi Ial2 NC 00602. Halichoerus grypus Man68 Zca Oro AY377297. Hydrurga leptonyx AY377296. Hydrurga leptonyx NC 008425. Hydrurga leptonyx AY377295. Ommatophoca rossii AY377294. Ommatophoca rossii Bas Bal AM7898. Spilogale putorius complete bcar24 AB29075. Meles meles anakuma 6 38 Ist 88 52 AB29076. Martes melampus AB29077. Enhydra lutris Ssu Car2 AY598506. AY598506S0 Alopex lagopus AH04073. SEG AY598506S Alopex lagopus CarBatEaredFox Band2and5 28 carwilddog DQ480508. Canis lupus isolate fro... AY598494. AY598494S0 Canis lupus DQ480502. Canis familiaris isolate... AH04072. SEG AY598494S Canis lupus DQ480507. Canis lupus isolate 2 fro... DQ480504. Canis lupus isolate fro... DQ48050. Canis familiaris isolate... AY656738. Canis familiaris isolate... AY729880. Canis familiaris AY656737. Canis familiaris isolate... NC 002008.4 Canis lupus familiaris 9 99 car2r Reverse Complement 6 Car2 Ppa286 57 35 Car8 CarLeop Ple85 48 Ple53 88 car84 75 lionf 35 43 87 DQ48049. Canis familiaris isolate... DQ480505. Canis lupus isolate fro... 58 DQ480492. Canis familiaris isolate... DQ480503. Canis lupus isolate fro... NC 008092. Canis lupus 86 DQ480506. Canis lupus isolate fro... car50copy 85 car85r Reverse Complement AY656739. Canis familiaris isolate... NC 008093. Canis latrans DQ4805. Canis latrans isolate 2 f... 99 DQ48050. Canis latrans isolate f... DQ480509. Canis latrans isolate f... Car79 45 car23 copy 78 Car53 Car8 2 car74r Reverse Complement 30 Car7 car63r Reverse Complement 7 Car57 Car56 27 Car car9r Reverse Complement 2 Car82 car45r Reverse Complement Car9ccf Car64 Car68 2 Car5 Car80 car90r Reverse Complement Car43 Car83 Car77 car44copy car55copy 48 car62 7 car66 27 car65copy 6 car60copy 46 34 Car75

car70r Reverse Complement 0 0 0 0 0 27 8 3 0 2 7 23 5 2 42 45 6 29 7 8 7 7 9 6 50 39 45 9 38 27 25 2 Car78 2 bfca57 Lse bfca53 bfca52 bfca54 Car73 bfca59 bfca55 bfca58 bfca02 bfca56 NC 00700. Felis catus Fma Fma 67Lse8 Fni serval 3 serval Fni4 AY598470. AY598470S0 Lynx canadensis AH04070. SEG AY598470S Lynx canadensis Car69 Cca2 Cca CarCaracal Car49 car88r Reverse Complement car93r Reverse Complement car54copy car47 car76r Reverse Complement Aju NC 00522. Acinonyx jubatus Car48 AY463959. Acinonyx jubatus car87r Reverse Complement 0 AF344830. Acinonyx jubatus car89r Reverse Complement 7 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 008450. Neofelis nebulosa Hhy40 Ccr34 Ccr236 Pli Vci babi Ial2 NC 00602. Halichoerus grypus Man68 Zca Oro AY377297. Hydrurga leptonyx AY377296. Hydrurga leptonyx NC 008425. Hydrurga leptonyx AY377295. Ommatophoca rossii AY377294. Ommatophoca rossii Bas Bal AM7898. Spilogale putorius complete bcar24 AB29075. Meles meles anakuma 6 25 6 48 9 97 57 35 46 88 38 97 95 46 70 75 30 34 70 52 99 97 50 33 4 23 39 88 35 57 Ist 43 88 52 AB29076. Martes melampus AB29077. Enhydra lutris Ssu Car2 78 2 AY598506. AY598506S0 Alopex lagopus AH04073. SEG AY598506S Alopex lagopus CarBatEaredFox Band2and5 87 30 45 7 27 99 Car79 86 Car53 carwilddog DQ480508. Canis lupus isolate fro... AY598494. AY598494S0 Canis lupus DQ480502. Canis familiaris isolate... AH04072. SEG AY598494S Canis lupus DQ480507. Canis lupus isolate 2 fro... DQ480504. Canis lupus isolate fro... DQ48050. Canis familiaris isolate... AY656738. Canis familiaris isolate... AY729880. Canis familiaris AY656737. Canis familiaris isolate... NC 002008.4 Canis lupus familiaris DQ48049. Canis familiaris isolate... DQ480505. Canis lupus isolate fro... DQ480492. Canis familiaris isolate... DQ480503. Canis lupus isolate fro... NC 008092. Canis lupus DQ480506. Canis lupus isolate fro... car50copy 85 car85r Reverse Complement AY656739. Canis familiaris isolate... NC 008093. Canis latrans DQ4805. Canis latrans isolate 2 f... DQ48050. Canis latrans isolate f... DQ480509. Canis latrans isolate f... Car8 car23 copy car74r Reverse Complement Car7 car63r Reverse Complement Car57 Car Car56 car9r Reverse Complement 2 Car82 car45r Reverse Complement Car64 Car68 2 Car5 48 7 27 6 Car80 Car9ccf car90r Reverse Complement Car43 Car83 Car77 car44copy car55copy car62 car66 46 34 58 28 car65copy car60copy Car75 43 DQ48049. Canis familiaris isolate... DQ480505. Canis lupus isolate fro... 58 DQ480492. Canis familiaris isolate... DQ480503. Canis lupus isolate fro... NC 008092. Canis lupus 86 DQ480506. Canis lupus isolate fro... car50 87 85car85R AY656739. Canis familiaris isolate... NC 008093. Canis latrans DQ4805. Canis latrans isolate 2 f... 99 DQ48050. Canis latrans isolate f... DQ480509. Canis latrans isolate f... Car79 car23 78 Car53 Car8 car74r Car7 car63r Car57 Car56 Car car9r Car82 car45r Car9ccf Car64 Car68 Car5 Car80 car90r Car43 Car83 Car77 car44copy car55copy car62 car66 car65copy car60copy Car75

Ongoing projects: phylogeny, phylogeography, population genetics and molecular ecology

Comparative phylogeography and demographic history of two South American procyonids: Nasua nasua and Procyon cancrivorus Mirian T. N. Tsuchiya-Jerep

N. nasua LBGM samples UCLA samples (Dr. Klaus Peter-Koepfli) Skin samples (MZUSP)

P. cancrivorus LBGM Samples Skin samples (MZUSP)

Data sets mtdna: 3 segments NADH Dehydrogenase subunit 5 (ND5) 750 bp Cytochrome b (Cytb) 40 bp Control region (D-loop) 300 bp Microsatellites Nasua nasua : development of a library and isolation of markers Procyon cancrivorus: heterologous transfer of markers from P. lotor.

Ln P(X D) Ln P(X D) Nasua nasua Procyon cancrivorus -800-900 -2000-2 -2200-2300 -2400-2500 0 2 4 6 8 0 Number of clusters (K ) -200-250 -300-350 -400-450 -500 0 2 4 6 8 0 Number of clusters (K )

Maned wolf conservation biology Integrating ecology, genetics, health sciences and education programs

Maned wolf population structure on a macrogeographic scale Manoel Rodrigues

Toninha Halei Pierre Mama Roni Piumhi Laís Jimy Guga Feminha Christy Ulna Roman Chew Lindi? Amadeu Thay Gambá Taís Sofia Melissa Cerveja Mel Chorão Roger Paquito Salete? Katrina Jerônimo Júnior Machinho Vitória

Molecular ecology of the Neotropical otter (Lontra longicaudis) Cristine Trinca A C B

DNA ext. cont. PCR Control PCR control Molecular ecology of the Neotropical otter (Lontra longicaudis) Cristine Trinca * *

Using scat DNA to predict a jaguar s coat color MCR NH 2 Extra-cellular Intra-cellular L T 99 94 L L E L 25 C D 2 S 90 Jaguar M 73 MCR - 5 S 7 Eizirik et al. 2003.

Genotyping of the jaguar MCR deletion

Genotypes for the 5 bp deletion in the jaguar MCR gene n* Genotypes 5 / 5 5 / + + / + Wild-type color 36 - - 36 Melanistic 0 9-46 9 36 *Geographic sampling: Mexico to southern Brazil; only unrelated animals were included. Fisher s Exact test: P<0.00

Using scat DNA to predict a jaguar s coat color. Expanded association study 2. Blind test with scat samples 3. Field test of scat samples

Conservation genetics of fragmented jaguar populations Modificado de Sanderson et al. (2002) e Zeller (2007)

Di Bitteti et al. 2003

Estimated number of individuals at each site: P. E. Morro do Diabo (SP): 9-5 (Cullen 2007) MS SP UE Porto Primavera (MS/SP): 0-20 (Crawshaw et al. 993) PR P. E. das Várzeas do Rio Ivinhema: 0 (Sana et al. 2006) Corredor verde (Brasil/Argentina/Paraguai): 25-53 (Paviolo et al. 2008)

Sampling UE Porto Primavera (SP/MS) (-6) n = 23 P. E. do Morro do Diabo (SP) (7) n = 8 P.E. das Várzeas Rio Ivinhema (MS) (8-9) n = 0 Corredor verde (Brasil/Arg/Paraguai) (-2) n = 8

Measures of diversity at 3 microsatellite loci in the four jaguar populations of the UPAF investigated in this study. Sample size (N), observed number of alleles (A), allelic richness (AR), observed (Ho) and expected (He) heterozygosities. Green Corridor (n= 8) Morro do Diabo (n= 8) Ivinhema (n= 0) Porto Primavera (n= 23) Locus N A AR H o H e 2 N A AR H o H e 2 N A AR H o H e 2 N A AR H o H e 2 FCA742 5 8 6.97 0.733 0.83 8 4 4.00 0.625 0.589 0 8 7.3 0.800 0.844 23 0 7.64 0.957 0.88 FCA723 6 5 4.00 0.500 0.708 8 2 2.00 0.250 0.232 0 2 2.00 0.400 0.5 23 6 4.7 0.696 0.707 FCA740 7 5 4.32 0.765 0.730 8 3 3.00 0.250 0.348 9 3 2.99 0.667 0.54 23 4 3.8 0.93 0.696 FCA44 8 4 3.79 0.444 0.639 8 2 2.00 0.25 0.25 0 4 3.77 0.700 0.656 23 4 3.79 0.696 0.660 FCA39 6 7 5.98 0.750 0.808 8 4 4.00 0.750 0.723 0 5 4.74 0.800 0.728 23 6 4.93 0.826 0.728 F98 7 3 2.86 0.588 0.57 8 3 3.00 0.625 0.482 0 4 3.97 0.800 0.77 23 3 2.83 0.609 0.552 F53 6 9 6.95 0.688 0.856 8 4 4.00.000 0.732 0 7 6.53 0.900 0.839 23 8 5.73 0.93 0.798 F24 7 7 5.50 0.824 0.76 8 4 4.00 0.750 0.74 0 6 6.53 0.800 0.8 23 7 5.72 0.826 0.803 F46 8 4 3.66 0.444 0.629 8 3 3.00 0.500 0.509 0 3 2.99 0.800 0.639 23 4 3.34 0.739 0.658 F85 4 0 7.55 0.643 0.860 8 3 3.00 0.625 0.607 9 5 4.88 0.778 0.708 22 6 4.99 0.88 0.769 F42 7 8 6.38 0.647 0.838 8 5 5.00 0.875 0.74 0 3 2.97 0.600 0.478 23 6 4.42 0.696 0.725 FCA453 3 5 4.8 0.538 0.792 8 3 3.00 0.750 0.66 0 3 2.80 0.400 0.350 23 5 4.25 0.783 0.723 FCA74 5 3 2.99 0.333 0.60 8.00-0.000 0 3 2.80 0.400 0.550 23 3 2.90 0.696 0.602 Overall 6.0 5.05 0.607 0.737 3.2 3.2 0.548 0.497 4.3 4.2 0.680 0.642 5.5 4.54 0.782 0.76 Allelic richness is calculated using minimum sample size of eight diploid individuals; 2 Unbiased gene diversity (corrected for sampling bias in FSTAT)

Relative frequencies Private alleles for each population: Morro do Diabo 0 Ivinhema 2 Porto Primavera 3 Corredor verde 8 0.4 0.3 0.2 0. 0 F53 60 64 76 80 84 88 92 96 200 204 Alleles Fig. 2 Histograms showing the distribution of the allele frequencies

Population structure: Identification of migrants and admixed individuals M M Corredor verde M.Diabo Ivinhema Porto Primavera AM

Genetic differentiation between populations: Table 4. Pairwise FST (above the diagonal) and RST values (below the diagonal) for the four jaguar populations of the UPAF. Green Corridor Morro do Diabo Ivinhema Porto Primavera Green Corridor - 0.98*** 0.22*** 0.048*** Morro do Diabo 0.2** - 0.20*** 0.073*** Ivinhema 0.08** -0.00-0.060*** Porto Primavera 0.036 * -0.007 0.024 - Significant values *P < 0.05, **P < 0.0 and ***P < 0.00

Effective population size estimates and their approximate confidence limits for each UPAF jaguar population based on two different methods (LDNE and ONESAMP). LDNE ( P crit = 0.05) ONESAMP (Prior = 2 -) N e Confidence limits (95%) N e Confidence limits (95%) Green Corridor 5.4 23.5-4 30.3 23.4-45.8 Morro do Diabo 4.6 2.3-2.8 7.8 6.8-9.8 Ivinhema 2.3 6.9-27. 0.3 9.2-2.5 Porto Primavera 3.5 0.4-8.0 2.7 9.4-26.6

Field data: None of the radio-colared individuals has dispersed among these study sites during recent monitoring Cullen 2007 Jaguars significantly avoid the use of human-modified areas, but some individuals do show long-distance movement across the fragmented landscape (Cullen 2007)

Conclusions -Surprising result given the geographic proximity of the field sites (ca. 69 km to 500 km) and the dispersal ability of this species - Molecular evidence for recent gene flow - Recent isolation (30-40 yrs, ca. 6-8 generations) - Small effective size and restricted gene flow among areas - Comparative data from other species Strong impact of humanindiced genetic drift in remnant jaguar populations

Neotropical felids: Genus Leopardus

Distributions of L. tigrinus and L. geoffroyi? Leopardus tigrinus L. colocolo Leopardus geoffroyi

Rio Grande do Sul State, Brazil E. Eizirik et al. 2006

Habitat use of L. tigrinus and L. geoffroyi in RS state Argentina N Uruguay 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 miles

% of prey in diet Dietary analysis of L. tigrinus e L. geoffroyi using stomach contents of roadkilled animals 30 25 Leopardus tigrinus Leopardus geoffroyi 20 5 0 5 0 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 20 67 93 78 99 99 99 80 96 39 CE0 Puma yagouaroundi Ne306 CE02 Puma yagouaroundi Ne306 OPN57 Oligoryzomys nigripes OPN87 Oligoryzomys nigripes OPN55 Oligoryzomys nigripes JR332 Oligoryzomys flavescens JR20 Oligoryzomys flavescens JR209 Oligoryzomys flavescens JR202 Oligoryzomys flavescens OPN60 Oligoryzomys nigripes JR244 Oligoryzomys flavescens CE32 L. geoffroyi Lgeof08 CE39 L. geoffroyi NE32 CE47 L. tigrinus blti22 EU095456 Oligoryzomys fulvescens PCE2 Deltamys kempi CE0 L. tigrinus NE32 CE20 L. tigrinus blti22 CE03 Leopardus geoffroyi Lge076 CE04 Leopardus geoffroyi Lge076 JR96 Sooretamys angouya 9790 Nectomys squamipes CE6 L. tigrinus blti00 89 68 45 99 87 86 78 65 92 59 99 58 CE63 L. tigrinus NE022 9735 Juliomys pictipes 9667 Juliomys pictipes 56 78 99 43 5 99 96 55 82 85 68 CE25 L. tigrinus blti079 CE65 L. tigrinus NE022 CE05 L. geoffroyi blge074 CE59 L. tigrinus blti069 JR 405 Calomys tener LBCE 5556 Calomys expulsus CRB 2732 Calomys expulsus LBCE 547 Calomys expulsus CE0 Leopardus geoffroyi Ne32 LBCE 548 Calomys expulsus CE66 L. tigrinus NE33 CE62 L. tigrinus NE022 JR395 Oxymycterus nasutus JR328 Akodon azarae JR329 Akodon azarae 9595 Akodon montensis PCE23 Oligoryzomys nigripes JR 407 Akodon montensis CE06 Leopardus geoffroyi Ne308 6844 Akodon paranaensis 6963 Akodon paranaensis CE09 P. yagouaroundi NE34 JR4 Akodon paranaensis 0.02

Acknowledgements Sandro L. Bonatto Thales R. Freitas Francisco M. Salzano Larissa Oliveira Flávio H. Rodrigues Dênis Sana Laury Cullen Tadeu Oliveira Cibele Indrusiak Ronaldo G. Morato Fernando Azevedo Fernanda Michalski Steve O Brien Warren Johnson Robert Wayne Klaus Koepfli Agostinho Antunes Bill Murphy Mark Springer Rogério de Paula Guilherme Mourão Rita Bianchi Fabio Mazim Cladinara R. Sarturi Support: CNPq, CAPES, FAPERGS, NIH, NSF, FNMA, CESP, WCS, WWF, CVRD, PUCRS Tatiane C. Trigo Taiana Haag Cristine S. Trinca Manoel F. Rodrigues Alexsandra Schneider Ezequiel Fabiano Eunice M. Matte Paulo B. Chaves Mirian Tsuchiya Fernanda P. Valdez Henrique V. Figueiró Flávia P. Tirelli Carlos Benhur Kasper Renata Bornholdt Anelisie S. Santos Taiz L. Simão Livia Lehugeur Camila Jaeger Vanessa Dias

Distributions of L. tigrinus, L. geoffroyi e L. colocolo? Leopardus tigrinus L. colocolo Leopardus geoffroyi

Sampling for genetic analyses (mtdna e STRs) Trigo et al. (2008) Mol. Ecol

Bayesian analysis of microsatellite genotypes Trigo et al. (2008) Mol. Ecol

mtdna Control Region blti30 blge06 blti33 blti60 blti34 blti0 blge46 blge79 blti7 blge Lco02 blti47 blti68 blti30 blti29 blti74 blti89 blti blti93 blti95 blti50 blti46 blti80 blti3 blti4 blti59 blti3 blti9 blti06 blge07 blti98 blti08 blti5 blti92 blti75 blge02 blge80 blti0 blti02 blti04 blti76 blti62 blti44 blti72 blge08 blti87 blti24 blti05 blti3 blti6 blge27 blti56 blti09 blti69 blge0 blti7 blti48 blti35 blti32 blti99 blti90 Ogu03 blge63 blge64 blge52 blti09 blge0 blti49 blti79 blge57 blge35 blge7 blge3g blti0 blge20 blge03 blti2 blge05 blge56 blge42 blge29 blge3 blge04 blge33 blge66 blge5 blge50 blge53 blge6 blge67 blge68 blge62 blti65 blge73 blge37 blge43 blge44 blge40 blge70 blge4 blge89 blge2 blge39 blco07 blco06 blco30 blco3 blti85 blti24 blti28 *

98 blti30 SP blge06 RS blti33 RS blti60 SP blti34 RS blti0 RS blge46 RS blge 79 RS blti7 SP blge RS Lco02 blti47 RS blti68 RS blti30 PAR blti29 PAR blti74 SP blti89 PR blti RS blti93 PR blti95 RS blti50 RS blti46 RS blti80 RS blti3 RS blti4 RS blti59 SP blti3 RS blti9 RS 62 blti06 RS blge07 RS blti98 RS blti08 RS blti5 RS blti92 PR blti75 SP blge02 RS blge80 RS blti0 RS blti02 RS blti04 RS blti76 SP blti62 SP blti44 SC blti72 MS blge08 RS blti87 SP blti24 RS blti05 RS blti3 PR blti6 SP blge27 RS blti56 SP blti09 MG blti69 RS blge0 RS blti7 RS blti48 RS blti35 RS blti32 RS blti99 RS blti90 PR 73 95 blge63 BOL blge64 BOL blge52 ARG blti09 RS blge0 RS blti49 RS blti79 RS blge57 URU blge35 RS blge7 RS blge3 RS blti0 RS 65 62 blge20 URU blge03 RS blti2 RS blge05 RS blge56 URU blge42 RS blge29 RS blge3 RS blge04 RS blge33 RS blge66 BOL blge5 ARG blge50 ARG blge53 ARG blge6 ARG blge67 BOL blge68 BOL blge62 BOL blti65 SP blge73 RS blge37 RS blge43 RS blge44 RS blge40 RS blge70 RS blge4 RS blge89 RS blge2 RS blge39 RS Ogu03 70 blco07 blco30 blti85 blti24 blti28 blco06 blco3 Brazilian populations of L. tigrinus may have mtdna lineages from three distinct species!

Haplotype network (mtdna) Leopardus tigrinus Clade Leopardus geoffroyi Clade 48 (+ 3) Trigo et al. (2008) Mol. Ecol

Hypothesis - recent demographic expansion of L. tigrinus - secondary contact with closely related species Subsequent steps: Increased use of multilocus data sets - Morpho-ecological analyses

Expanded sampling 0 o MA CE PI PE SE N 30 ºS BOL ARG PAR MS PR GO SP SC MG RJ ES Central Region Northeastern Region A CH URU RS Southern Region Leopardus tigrinus Leopardus geoffroyi Leopardus colocolo 60 ºW

mtdna haplotype networks L. geoffroyi clade L. tigrinus clade L. guigna L. pardalis L. wiedii L. colocolo clade L. tigrinus (C, NE Brasil)

Haplotype network (intron 2, PLP gene, X chromosome) L. geoffroyi L. guigna L. colocolo L. pardalis + L. wiedii

Haplotype network (intron 2, PLP gene, X chromosome) L. tigrinus (S, SE Brasil) L. tigrinus (C, NE Brasil) L. guigna L. pardalis + L. wiedii

Haplotype network (intron 2, ZFY and SMCY3 genes, Y chromosome) L. tigrinus (S, SE Brasil) L. geoffroyi L. tigrinus (C, NE Brasil) L. colocolo L. wiedii

Bayesian analysis of microsatellites L. geoffroyi L. tigrinus (S, SE Brasil) L. tigrinus (C, NE Brasil) L. colocolo

q Detailed analyses of the hybrid zone: 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0. 0 6 6 2 26 3 36 4 46 5 56 6 66 7 76 8 86 9 individuals rank

Score (76.9%) Canonic analysis based on 24 body measurements 6.0 2.0 GM GF TM TF -2.0-6.0-4.0 -.3.3 4.0 Score 2 (7.7%)

Correspondence analysis between genetic and morphological categories GPG LGE LTI TPT HIB GPI TPI

Individual case studies: - Morphological identification - mtdna haplotype - Y chromosome haplotype - PLP (X chrom.) haplotypes - microsatellite-based assignment (quantification of genome composition)

blge46 Canela, RS? Microsatellites 3rd Generation PLP: L. geoffroyi Zfy L. tigrinus mtdna: L. tigrinus

blti79 Guaíba, RS Microsatellites st Generation PLP: L. tigrinus + L. geoffroyi mtdna: L. geoffroyi