The Use of Molecular Tools in Ecological Studies

International Workshop on “Applied Ecology and Human Dimensions in Biological Conservation” – Biota FAPESP - 2009

Eduardo Eizirik

Faculdade de Biociências, PUCRS, Brasil Instituto Pró-Carnívoros, Brasil Laboratory of Genomic Diversity, NIH, USA Molecular Markers in Conservation Biology 1. 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 …

1. 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 10. 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) 100 bPon32 (sangue de Panthera onca) bPon56 (fezes de Panthera onca em DET) P3-16 (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) P31-1 (pêlos de Panthera onca) F2-636 (fezes de felino grande) 99 Puma concolor (sangue) L.V.(fezes de felino grande) F57-1C (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 12 Car78 21 bFca57 30 bFca53 bFca52 34 57 bFca54

46 Car73 bFca59 bFca55 70 52 bFca58 39 bFca02 car70R Reverse Complement 91 bFca56 25 NC 001700.1| Felis catus

18 Fma 97 Fma11 12 Fni Car78 100 Fni14 1 70 serval 3 100 serval 1 21 Lse bFca57 67Lse18

7 100 AY598470.1|AY598470S01 Lynx canadensis 1 AH014070.1|SEG AY598470S Lynx canadensis 7 Car69 Cca21 100 30 bFca53 Cca1 88 52 CarCaracal Car49 car88R Reverse Complement 97 car93R Reverse Complement bFca52 46 car54copy 34 50 car47 33 car76R Reverse Complement 0 14 Aju 57 bFca54 NC 005212.1| Acinonyx jubatus 23 Car48 AY463959.1| Acinonyx jubatus 1 car87R Reverse Complement 0 AF344830.1| Acinonyx jubatus Car73 1 46 1 car89R Reverse Complement 17 car94R Reverse Complement

99 car2R Reverse Complement 0 61 Car2 bFca59 Ppa286 57 Car8ccfAndBrazil

91 35 CarLeop Ple185 48 Ple153 bFca55 car84copy Reverse Complement 88 75 lionF 0 Cfe1 52 car52 3 70 bFca58 NC 008450.1| Neofelis nebulosa 5 Vci 7 38 bAbi 97 Ial2 Rme 39 bFca02 1 19 Hja 2 Hpa1

8 100 AY598506.1|AY598506S01 Alopex lagopus AH014073.1|SEG AY598506S Alopex lagopus 23 CarBatEaredFox carWildDog 45 B11and12and15 DQ480508.1| Canis lupus isolate 1 fro...

45 car23 copy 78 Car53 Car81 21 car74R Reverse Complement 30 Car71 car63R Reverse Complement 17 Car57 Car56 27 Car11 car91R Reverse Complement 12 Car82 car45R Reverse Complement Car9ccf Car64 Car68 12 Car15 Car80 car90R Reverse Complement Car43 Car83 Car77 car44copy car55copy IUCN Cat Specialist Group 48 car62 17 car66 27 car65copy 16 car60copy 46 34 Car75

0,02 25 car70R Reverse Complement 12 Car78 21 bFca57 30 bFca53 bFca52 34 57 bFca54

46 Car73 bFca59 bFca55 70 52 bFca58 39 bFca02 91 bFca56 NC 001700.1| Felis catus

18 Fma 97 Fma11 Fni 100 Fni14 1 70 serval 3 100 serval 1 Lse 67Lse18

7 100 AY598470.1|AY598470S01 Lynx canadensis 1 AH014070.1|SEG AY598470S Lynx canadensis 7 Car69 Cca21 100 Cca1 88 52 CarCaracal Car49 car88R Reverse Complement 97 car93R Reverse Complement 46 car54copy 50 car47 33 car76R Reverse Complement

0 14 Aju NC 005212.1| Acinonyx jubatus 23 Car48 AY463959.1| Acinonyx jubatus 1 car87R Reverse Complement 0 AF344830.1| Acinonyx jubatus 1 1 car89R Reverse Complement 17 car94R Reverse Complement

99 car2R Reverse Complement 0 61 Car2 Ppa286 57 Car8ccfAndBrazil

91 35 CarLeop Ple185

48 Ple153 car84copy Reverse Complement 88 75 lionF 0 Cfe1 car52 3 NC 008450.1| Neofelis nebulosa 5 Vci 7 38 bAbi 97 Ial2 Rme 1 19 Hja 2 Hpa1

2 Pcr Hbr1 1 39 Hhy40 27 Ccr34 Car69 25 88 Ccr236 Phe Pli 61 NC 001602.1| Halichoerus grypus Cca21 42 Zca Oro 100 AY377297.1| Hydrurga leptonyx 27 100 AY377296.1| Hydrurga leptonyx NC 008425.1| Hydrurga leptonyx Cca1 45 Man68 88 AY377295.1| Ommatophoca rossii 50 100 AY377294.1| Ommatophoca rossii 95 Bal 52 Caracal 16 Bas 7 AM711898.1| Spilogale putorius complete 0 bCar24 AB291076.1| Martes melampus 1 29 AB291075.1| Meles meles anakuma AB291077.1| Enhydra lutris 0 Ist Ssu 10 Car12

8 100 AY598506.1|AY598506S01 Alopex lagopus AH014073.1|SEG AY598506S Alopex lagopus 23 CarBatEaredFox carWildDog 45 B11and12and15 DQ480508.1| Canis lupus isolate 1 fro...

45 car23 copy 78 Car53 Car81 21 car74R Reverse Complement 30 Car71 car63R Reverse Complement 17 Car57 Car56 27 Car11 car91R Reverse Complement 12 Car82 car45R Reverse Complement Car9ccf Car64 Car68 12 Car15 Car80 car90R Reverse Complement Car43 Car83 Car77 car44copy car55copy 48 car62 17 car66 27 car65copy 16 car60copy 46 34 Car75

0,02 25 car70R Reverse Complement 12 Car78 21 bFca57 30 bFca53 bFca52 34 57 bFca54

46 Car73 bFca59 bFca55 70 52 bFca58 39 bFca02 91 bFca56 NC 001700.1| Felis catus

18 Fma 97 Fma11 Fni 100 Fni14 1 70 serval 3 100 serval 1 Lse 67Lse18

0 14 Aju NC 005212.1| Acinonyx jubatus 23 Car48 AY463959.1| Acinonyx jubatus 1 car87R Reverse Complement 0 AF344830.1| Acinonyx jubatus 1 1 car89R Reverse Complement 17 car94R Reverse Complement Car49 99 car2R Reverse Complement 0 61 Car2 Ppa286 57 Car8ccfAndBrazil 91 CarLeop car88R Reverse Complement 35 Ple185 48 Ple153 97 car84copy Reverse Complement 88 75 lionF 0 car93R Reverse Complement Cfe1 car52 3 NC 008450.1| Neofelis nebulosa 46 5 Vci 7 38 bAbi car54copy 97 Ial2 Rme 1 Hja 50 19 2 Hpa1 car47 2 Pcr Hbr1 1 39 Hhy40 27 Ccr34 33 25 88 Ccr236 car76R Reverse Complement Phe Pli

8 100 AY598506.1|AY598506S01 Alopex lagopus AH014073.1|SEG AY598506S Alopex lagopus car87R Reverse Complement 23 CarBatEaredFox carWildDog 0 45 B11and12and15 DQ480508.1| Canis lupus isolate 1 fro... AF344830.1| Acinonyx jubatus 61 AY598494.1|AY598494S01 Canis lupus DQ480502.1| Canis familiaris isolate ... 1 AH014072.1|SEG AY598494S Canis lupus DQ480507.1| Canis lupus isolate 2 fro... 38 DQ480504.1| Canis lupus isolate 1 fro... car89R Reverse Complement DQ480501.1| Canis familiaris isolate ... 1 AY656738.1| Canis familiaris isolate ... 28 AY729880.1| Canis familiaris AY656737.1| Canis familiaris isolate ... NC 002008.4| Canis lupus familiaris 17 car94R Reverse Complement 35 DQ480491.1| Canis familiaris isolate ... DQ480505.1| Canis lupus isolate 1 fro... 58 DQ480492.1| Canis familiaris isolate ... DQ480503.1| Canis lupus isolate 1 fro... NC 008092.1| Canis lupus 86 DQ480506.1| Canis lupus isolate 1 fro... car50copy 85 87 car85R Reverse Complement AY656739.1| Canis familiaris isolate ... NC 008093.1| Canis latrans DQ480511.1| Canis latrans isolate 2 f... 99 DQ480510.1| Canis latrans isolate 1 f... 43 DQ480509.1| Canis latrans isolate 1 f... Car79

45 car23 copy 78 Car53 Car81 21 car74R Reverse Complement 30 Car71 car63R Reverse Complement 17 Car57 Car56 27 Car11 car91R Reverse Complement 12 Car82 car45R Reverse Complement Car9ccf Car64 Car68 12 Car15 Car80 car90R Reverse Complement Car43 Car83 Car77 car44copy car55copy 48 car62 17 car66 27 car65copy 16 car60copy 46 34 Car75

0,02 25 car70R Reverse Complement 12 Car78 21 bFca57 30 bFca53 bFca52 34 57 bFca54

46 Car73 bFca59 bFca55 70 52 bFca58 39 bFca02 91 bFca56 NC 001700.1| Felis catus

18 Fma 97 Fma11 Fni 100 Fni14 1 70 serval 3 100 serval 1 Lse 67Lse18

0 14 Aju NC 005212.1| Acinonyx jubatus 23 Car48 AY463959.1| Acinonyx jubatus 1 car87R Reverse Complement 0 AF344830.1| Acinonyx jubatus 1 1 car89R Reverse Complement 17 car94R Reverse Complement

99 car2R Reverse Complement 0 61 Car2 Ppa286 57 Car8ccfAndBrazil

91 35 CarLeop Ple185

48 Ple153 car84copy Reverse Complement 88 75 lionF 0 Cfe1 car52 3 NC 008450.1| Neofelis nebulosa 5 Vci 7 38 bAbi 97 Ial2 Rme 1 19 Hja car2R Reverse Complement 2 Hpa1 99

2 Pcr Hbr1 1 39 Hhy40 27 Car2 Ccr34 25 61 88 Ccr236 Phe Pli 61 NC 001602.1| Halichoerus grypus Ppa286 42 Zca Oro AY377297.1| Hydrurga leptonyx 27 100 AY377296.1| Hydrurga leptonyx NC 008425.1| Hydrurga leptonyx 57 Car8 45 Man68 AY377295.1| Ommatophoca rossii 50 100 AY377294.1| Ommatophoca rossii 95 Bal CarLeop 16 Bas 91 35 7 AM711898.1| Spilogale putorius complete 0 bCar24 AB291076.1| Martes melampus 1 29 AB291075.1| Meles meles anakuma Ple185 AB291077.1| Enhydra lutris 0 Ist Ssu 10 Car12 Ple153 8 100 AY598506.1|AY598506S01 Alopex lagopus 48 AH014073.1|SEG AY598506S Alopex lagopus 23 CarBatEaredFox carWildDog 45 B11and12and15 car84 DQ480508.1| Canis lupus isolate 1 fro... 88 61 AY598494.1|AY598494S01 Canis lupus DQ480502.1| Canis familiaris isolate ... AH014072.1|SEG AY598494S Canis lupus 75 lionF DQ480507.1| Canis lupus isolate 2 fro... 38 DQ480504.1| Canis lupus isolate 1 fro... DQ480501.1| Canis familiaris isolate ... AY656738.1| Canis familiaris isolate ... 28 AY729880.1| Canis familiaris AY656737.1| Canis familiaris isolate ... NC 002008.4| Canis lupus familiaris

45 car23 copy 78 Car53 Car81 21 car74R Reverse Complement 30 Car71 car63R Reverse Complement 17 Car57 Car56 27 Car11 car91R Reverse Complement 12 Car82 car45R Reverse Complement Car9ccf Car64 Car68 12 Car15 Car80 car90R Reverse Complement Car43 Car83 Car77 car44copy car55copy 48 car62 17 car66 27 car65copy 16 car60copy 46 34 Car75

0,02 25 car70R Reverse Complement 12 Car78 21 bFca57 30 bFca53 bFca52 34 57 bFca54

46 Car73 bFca59 bFca55 70 52 bFca58 39 bFca02 91 bFca56 NC 001700.1| Felis catus

18 Fma 97 Fma11 Fni 100 Fni14 1 70 serval 3 100 serval 1 DQ480491.1| Canis familiaris isolate ... Lse 67Lse18 7 100 AY598470.1|AY598470S01 Lynx canadensis DQ480505.1| Canis lupus isolate 1 fro... 1 AH014070.1|SEG AY598470S Lynx canadensis 58 7 Car69 Cca21 100 DQ480492.1| Canis familiaris isolate ... Cca1 88 52 CarCaracal Car49 DQ480503.1| Canis lupus isolate 1 fro... car88R Reverse Complement 97 car93R Reverse Complement 46 car54copy NC 008092.1| Canis lupus 50 car47 33 86 car76R Reverse Complement

0 14 Aju DQ480506.1| Canis lupus isolate 1 fro... NC 005212.1| Acinonyx jubatus 23 Car48 AY463959.1| Acinonyx jubatus car50 1 car87R Reverse Complement 0 AF344830.1| Acinonyx jubatus 1 car89R Reverse Complement 85car85R 1 17 car94R Reverse Complement 87

99 car2R Reverse Complement 0 Car2 AY656739.1| Canis familiaris isolate ... 61 Ppa286 57 Car8ccfAndBrazil CarLeop NC 008093.1| Canis latrans 91 35 Ple185 48 Ple153 DQ480511.1| Canis latrans isolate 2 f... car84copy Reverse Complement 88 75 lionF 0 Cfe1 99 DQ480510.1| Canis latrans isolate 1 f... car52 3 NC 008450.1| Neofelis nebulosa 43 5 Vci 7 DQ480509.1| Canis latrans isolate 1 f... 38 bAbi 97 Ial2 Rme 1 Car79 19 Hja 2 Hpa1 2 Pcr car23 Hbr1 1 39 Hhy40 27 Ccr34 25 78 Car53 88 Ccr236 Phe Pli Car81 61 NC 001602.1| Halichoerus grypus 42 Zca Oro car74R AY377297.1| Hydrurga leptonyx 27 100 AY377296.1| Hydrurga leptonyx NC 008425.1| Hydrurga leptonyx Car71 45 Man68 AY377295.1| Ommatophoca rossii 50 100 AY377294.1| Ommatophoca rossii car63R 95 Bal 16 Bas 7 AM711898.1| Spilogale putorius complete Car57 0 bCar24 AB291076.1| Martes melampus 1 29 AB291075.1| Meles meles anakuma Car56 AB291077.1| Enhydra lutris 0 Ist Ssu Car11 10 Car12

8 100 AY598506.1|AY598506S01 Alopex lagopus AH014073.1|SEG AY598506S Alopex lagopus car91R 23 CarBatEaredFox carWildDog 45 B11and12and15 Car82 DQ480508.1| Canis lupus isolate 1 fro...

61 AY598494.1|AY598494S01 Canis lupus DQ480502.1| Canis familiaris isolate ... car45R AH014072.1|SEG AY598494S Canis lupus DQ480507.1| Canis lupus isolate 2 fro... 38 Car9ccf DQ480504.1| Canis lupus isolate 1 fro... DQ480501.1| Canis familiaris isolate ... AY656738.1| Canis familiaris isolate ... 28 Car64 AY729880.1| Canis familiaris AY656737.1| Canis familiaris isolate ... NC 002008.4| Canis lupus familiaris Car68 35 DQ480491.1| Canis familiaris isolate ... DQ480505.1| Canis lupus isolate 1 fro... 58 DQ480492.1| Canis familiaris isolate ... Car15 DQ480503.1| Canis lupus isolate 1 fro... NC 008092.1| Canis lupus 86 DQ480506.1| Canis lupus isolate 1 fro... Car80 car50copy 85 87 car85R Reverse Complement AY656739.1| Canis familiaris isolate ... car90R NC 008093.1| Canis latrans DQ480511.1| Canis latrans isolate 2 f... 99 DQ480510.1| Canis latrans isolate 1 f... Car43 43 DQ480509.1| Canis latrans isolate 1 f... Car79 45 car23 copy Car83 78 Car53 Car81 21 car74R Reverse Complement Car77 30 Car71 car63R Reverse Complement 17 Car57 car44copy Car56 27 Car11 car91R Reverse Complement car55copy 12 Car82 car45R Reverse Complement Car9ccf car62 Car64 Car68 12 Car15 car66 Car80 car90R Reverse Complement Car43 car65copy Car83 Car77 car44copy car60copy car55copy 48 car62 17 car66 Car75 27 car65copy 16 car60copy 46 34 Car75

0,02 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) – 1140 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. Nasua nasua Procyon cancrivorus

-1800

-1900 -1200 -2000 -1250 -2100 -1300 -2200 LnP(X|D) -1350

-2300 LnP(X|D) -1400 -2400 -1450 -2500 0 2 4 6 8 10 -1500 Number of clusters (K ) 0 2 4 6 8 10 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 Pierre Mama Halei Roni

Piumhi Ulna Jimy Guga Feminha Christy Roman Laís

Amadeu Thay Gambá Chew Lindi ?

Taís Sofia Melissa Cerveja

Mel Chorão Roger

Katrina Jerônimo Júnior Paquito Salete ?

Vitória Machinho Molecular ecology of the Neotropical otter (Lontra longicaudis) Cristine Trinca

A C

B Molecular ecology of the Neotropical otter ( otter Neotropical the of ecology Molecular

DNA ext. cont.

PCR Control PCR control Lontra longicaudis Lontra * Cristine Trinca * ) Using scat DNA to predict a jaguar’s coat color MC1R

NH 2 Extra-cellular

Intra-cellular L T

100 L L 99 L D 121 94 E 125 C

S 90

Jaguar

M 73 MC1R - 15 S 71 Eizirik et al. 2003. Genotyping of the jaguar MC1R deletion Genotypes for the 15 bp deletion in the jaguar MC1R gene

n* Genotypes 15 / 15 15 / + + / +

Wild-type color 36 - - 36 Melanistic 10 1 9 -

46 1 9 36 *Geographic sampling: Mexico to southern Brazil; only unrelated animals were included.

Fisher’s Exact test: P<0.001 Using scat DNA to predict a jaguar’s coat color

1. 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): 9-15 (Cullen 2007) SP MS

UE Porto Primavera (MS/SP): 10-20 (Crawshaw et al. 1993) PR

P. E. das Várzeas do Rio Ivinhema: 10 (Sana et al. 2006)

Corredor verde (Brasil/Argentina/Paraguai): 25-53 (Paviolo et al. 2008) Sampling

UE Porto Primavera (SP/MS) (1-6) → n = 23

P. E. do Morro do Diabo (SP) (7) → n = 8

P.E. das Várzeas Rio Ivinhema (MS) (8-9) → n = 10

Corredor verde (Brasil/Arg/Paraguai) (11-21) → n = 18 Measures of diversity at 13 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= 18) Morro do Diabo (n= 8) Ivinhema (n= 10) Porto Primavera (n= 23)

1 2 1 2 1 2 1 2 Locus N A AR Ho He N A AR Ho He N A AR Ho He N A AR Ho He FCA742 15 8 6.97 0.733 0.831 8 4 4.00 0.625 0.589 10 8 7.31 0.800 0.844 23 10 7.64 0.957 0.881 FCA723 16 5 4.00 0.500 0.708 8 2 2.00 0.250 0.232 10 2 2.00 0.400 0.511 23 6 4.71 0.696 0.707 FCA740 17 5 4.32 0.765 0.730 8 3 3.00 0.250 0.348 9 3 2.99 0.667 0.514 23 4 3.81 0.913 0.696 FCA441 18 4 3.79 0.444 0.639 8 2 2.00 0.125 0.125 10 4 3.77 0.700 0.656 23 4 3.79 0.696 0.660 FCA391 16 7 5.98 0.750 0.808 8 4 4.00 0.750 0.723 10 5 4.74 0.800 0.728 23 6 4.93 0.826 0.728 F98 17 3 2.86 0.588 0.517 8 3 3.00 0.625 0.482 10 4 3.97 0.800 0.717 23 3 2.83 0.609 0.552 F53 16 9 6.95 0.688 0.856 8 4 4.00 1.000 0.732 10 7 6.53 0.900 0.839 23 8 5.73 0.913 0.798 F124 17 7 5.50 0.824 0.761 8 4 4.00 0.750 0.741 10 6 6.53 0.800 0.811 23 7 5.72 0.826 0.803 F146 18 4 3.66 0.444 0.629 8 3 3.00 0.500 0.509 10 3 2.99 0.800 0.639 23 4 3.34 0.739 0.658 F85 14 10 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 0.818 0.769 F42 17 8 6.38 0.647 0.838 8 5 5.00 0.875 0.714 10 3 2.97 0.600 0.478 23 6 4.42 0.696 0.725 FCA453 13 5 4.81 0.538 0.792 8 3 3.00 0.750 0.661 10 3 2.80 0.400 0.350 23 5 4.25 0.783 0.723 FCA741 15 3 2.99 0.333 0.610 8 1 1.00 - 0.000 10 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 0.716

1Allelic richness is calculated using minimum sample size of eight diploid individuals; 2 Unbiased gene diversity (corrected for sampling bias in FSTAT) Private alleles for each population:

Morro do Diabo→ 0 Ivinhema→ 2 Porto Primavera→ 3 Corredor verde→ 18

F53 0.4 0.3 0.2 0.1

RelativeRelative frequencies frequencies 160 164 176 180 184 188 192 196 200 204

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 - 0.198*** 0.122*** 0.048*** Morro do Diabo 0.112** - 0.120*** 0.073*** Ivinhema 0.081** -0.010 - 0.060*** Porto Primavera 0.036 * -0.007 0.024 -

Significant values *P < 0.05, **P < 0.01 and ***P < 0.001 Effective population size estimates and their approximate confidence limits for each UPAF jaguar population based on two different methods (LDNE and ONESAMP).

LDNE ( Pcrit= 0.05) ONESAMP (Prior = 2 -100) N Confidence limits (95%) N Confidence limits (95%) e e Green Corridor 51.4 23.5 - 11004 30.3 23.4 - 45.8 Morro do Diabo 4.6 2.3 - 12.8 7.8 6.8 - 9.8 Ivinhema 12.3 6.9 - 27.1 10.3 9.2 - 12.5 Porto Primavera 13.5 10.4 - 18.0 21.7 19.4 - 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 human- indiced genetic drift in remnant jaguar populations Neotropical felids: Genus Leopardus Distributions of L. tigrinus and L. geoffroyi

Leopardus tigrinus

Leopardus geoffroyi L. colocolo Rio Grande do Sul State, Brazil

E. Eizirik et al. 2006

Habitat use of L. tigrinus and L. geoffroyi in RS state

Argentina

Gramineous Steppe Park Steppe Arboreal Steppe Steppical Savannah Semidecidual Stational Forest Uruguay Decidual Stational Forest Atlantic OceanMixed Ombrophilous Forest Dense Ombrophilous Forest N Pioneer Formations Ecological Tension Area

100 miles Dietary analysis of L. tigrinus e L. geoffroyi using stomach contents of roadkilled animals

30 Leopardus tigrinus 25 Leopardus geoffroyi

10 % of prey in diet in prey of %

100 LBCE 1547|Calomys expulsus 85 CE011|Leopardus geoffroyi|Ne312 68 LBCE 1548|Calomys expulsus

100 JR328|Akodon azarae 93 JR329|Akodon azarae

96 6963|Akodon paranaensis 55 CE09|P. yagouaroundi|NE314 82 JR411|Akodon paranaensis

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

Leopardus tigrinus

Leopardus geoffroyi L. colocolo Sampling for genetic analyses (mtDNA e STRs)

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

Trigo et al. (2008) Mol. Ecol bLti301 bLge06 bLti133 bLti60 bLti134 bLti110 bLge46 bLge79 mtDNA Control Region bLti71 bLge11 Lco02 bLti47 bLti68 bLti30 bLti29 bLti74 bLti89 bLti100 bLti93 bLti95 bLti50 bLti46 bLti80 bLti113 bLti114 bLti59 bLti131 bLti119 bLti106 bLge07 bLti98 bLti108 bLti51 bLti92 bLti75 bLge02 bLge80 bLti10 bLti102 bLti04 bLti76 bLti62 bLti44 bLti72 bLge08 bLti87 bLti124 bLti05 bLti31 bLti61 bLge27 bLti56 bLti109 bLti69 bLge01 bLti117 bLti48 bLti135 bLti132 bLti99 bLti90 Ogu03 bLge63 bLge64 bLge52 * bLti09 bLge10 bLti49 bLti79 bLge57 bLge35 bLge71 bLge13g bLti01 bLge20 bLge03 bLti121 bLge05 bLge56 bLge42 bLge29 bLge31 bLge04 bLge33 bLge66 bLge51 bLge50 bLge53 bLge61 bLge67 bLge68 bLge62 bLti65 bLge73 bLge37 bLge43 bLge44 bLge40 bLge70 bLge41 bLge89 bLge12 bLge39 bLco07 bLco06 bLco30 bLco13 bLti85 bLti24 bLti28 bLti301 SP bLge06 RS Ogu03 bLti133 RS bLge63 BOL bLti60 SP 73 bLti134 RS bLge64 BOL bLti110 RS 95 bLge52 ARG bLge46 RS bLge 79 RS bLti09 RS bLti71 SP bLge10 RS bLge11 RS Lco02 bLti49 RS bLti47 RS bLti79 RS bLti68 RS bLge57 URU bLti30 PAR bLti29 PAR bLge35 RS bLti74 SP bLge71 RS bLti89 PR bLge13 RS bLti100 RS bLti93 PR bLti01 RS bLti95 RS bLge20 URU bLti50 RS bLge03 RS bLti46 RS bLti80 RS bLti121 RS bLti113 RS bLge05 RS bLti114 RS bLge56 URU bLti59 SP 65 bLco07 bLti131 RS bLge42 RS bLco06 bLti119 RS bLge29 RS 70 62 bLti106 RS bLco30 bLge07 RS bLge31 RS bLti98 RS bLge04 RS bLco13 bLti108 RS bLge33 RS bLti51 RS bLti85 bLge66 BOL bLti92 PR bLti24 bLti75 SP bLge51 ARG bLge02 RS bLge50 ARG bLti28 bLge80 RS bLti10 RS bLge53 ARG bLti102 RS bLge61 ARG bLti04 RS bLge67 BOL bLti76 SP bLti62 SP bLge68 BOL bLti44 SC bLge62 BOL bLti72 MS bLti65 SP bLge08 RS bLti87 SP bLge73 RS bLti124 RS bLge37 RS bLti05 RS bLti31 PR bLge43 RS bLti61 SP bLge44 RS bLge27 RS 62 bLge40 RS bLti56 SP bLti109 MG bLge70 RS Brazilian populations of L. bLti69 RS bLge41 RS bLge01 RS bLge89 RS tigrinus may have mtDNA bLti117 RS bLti48 RS bLge12 RS 98 bLti135 RS bLge39 RS lineages from three bLti132 RS bLti99 RS distinct species! bLti90 PR Haplotype network (mtDNA)

Leopardus tigrinus Clade Leopardus geoffroyi Clade

48 (+ 13)

Trigo et al. (2008) Mol. Ecol Hypothesis

- recent demographic expansion of L. tigrinus - secondary contact with closely related species

Subsequent steps: Increased use of multi- locus data sets

- Morpho-ecological analyses Expanded sampling

0 o

MA CE

PI PE

BOL GO MG NortheastNortheastern MS ES Region SP PAR RJ PR Central ARG SC N 30 ºS Region RS

CH URU SouthSouthern A Region

Leopardus tigrinus Leopardus geoffroyi Leopardus colocolo

60 ºW mtDNA haplotype networks

L. geoffroyi clade

L. tigrinus clade

L. guigna

L. pardalis L. tigrinus (C, NE Brasil) L. wiedii L. colocolo clade Haplotype network (intron 2, PLP1 gene, X chromosome)

L. geoffroyi

L. guigna L. colocolo

L. pardalis + L. wiedii Haplotype network (intron 2, PLP1 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. tigrinus (C, NE Brasil) L. geoffroyi

L. colocolo

L. wiedii Bayesian analysis of microsatellites

L. geoffroyi L. colocolo L. tigrinus L. tigrinus (S, SE Brasil) (C, NE Brasil) Detailed analyses of the hybrid zone:

1 0.9 0.8 0.7 0.6

0.5 q1 0.4 0.3 0.2 0.1 0 1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 individuals rank Canonic analysis based on 24 body measurements

6.0

GM GF TM 2.0 TF

-2.0 Score 1 (76.9%)

-6.0 -4.0 -1.3 1.3 4.0 Score 2 (17.7%) Correspondence analysis between genetic and morphological categories

GPG TPT LGE LTI

HIB

GPI TPI Individual case studies:

- Morphological identification - mtDNA haplotype - Y chromosome haplotype - PLP1 (X chrom.) haplotypes - microsatellite-based assignment (quantification of genome composition) bLge46 Canela, RS ?

Microsatellites  3rd Generation PLP1:  L. geoffroyi Zfy  L. tigrinus mtDNA:  L. tigrinus bLti79 Guaíba, RS

Microsatellites  1st Generation PLP1:  L. tigrinus + L. geoffroyi mtDNA:  L. geoffroyi