1. Genetic Diversity and Fitness in Black-Footed Ferrets Before and During a Bottleneck S.M. Wisely, S.W. Buskirk, M.A. Fleming, D.B. Mc Donald, and E.A. Ostrander Presented by: Melanie Crawford and Julie Bernard

2. Background Information Black-footed ferret (Mustela nigripes) is an endangered N. American carnivore Population decline due to habitat loss and land conversion during 20 th century Underwent bottleneck in mid-1980’s

3. Background Continued 1985 last wild population (N=40) Epizootics of canine distemper and sylvatic plague 18 individuals captured for breeding By 1986 no individuals living in the wild and 7 captive adults

4. Objectives To compare genetic diversity before versus during the bottleneck To compare the last wild population to two historical populations To determine whether fitness changes accompanied population changes

5. Objectives continued To compare the genetic diversity in black-footed ferrets to that of two sibling species, the steppe polecat (Mustela eversmanni) and the European polecat (Mustela putorius) Mustela eversmanni Mustela putorius

6. Prediction and Past Info Population genetic theory predicts and some empirical data suggests that genetic variability will decline, and individual fitness and adaptability will be lowered. Previous investigations of genetic diversity in black-footed ferrets found little or no allozyme variation Conservation biologists usually study populations after they become threatened and small

7. This study makes use of museum specimens to examine the bottleneck, as well as, historical genetic diversity More accurate because it compares current diversity with past diversity

8. Estimated genetic diversity in two sibling taxa with different population histories: - an undisturbed population of the steppe polecat (Mustela eversmanni) - a bottlenecked population of the European polecat (Mustela putorius) Used to determine if interspecific comparisons of genetic diversity would accurately reflect the bottleneck in black-footed ferrets

9. Measured litter size in pre- and post- bottleneck black-footed ferrets to measure fitness The data from this experiment provide a test of a fundamental tenet of conservation biology, that severe bottlenecks lead to fitness losses that increase the likelihood of extinction

10. Materials and Methods Samples Tissue from 20 black-footed ferrets collected between 1891 and 1985 -Trego County, KS 1891 (n=4; dried skeletal muscle) -Mellette County, SD 1972 (n=4; frozen skeletal muscle) -Meeteetse, WY 1983-1985 (12 individuals; frozen kidney, heart, skeletal muscle)

11. Samples continued Steppe polecats (5 individuals; frozen skeletal muscle) -from a single location near Ba Daer Hu, Inner Mongolia European polecats (6 individuals; skeletal muscle in ethanol) -from a single contemporary population in west- central England

12. DNA Analysis DNA Extracted Amplified 24 Microsatellite loci

13. Statistical Analysis Deviations from HWE in pre-bottleneck WY of black-footed ferrets using exact test of HWE Linkage Disequilibrium between pairs of loci in the WY population using an exact test of P Inbreeding within sub-populations of black- footed ferrets and genetic structure among subpopulations

14. Statistical Analysis continued Differences in genetic diversity among five groups of ferrets: steppe polecat, European polecat, KS, SD, and WY Used three measures of genetic diversity -the number of alleles per locus averaged across loci (A) -the percent polymorphic loci (P o ) -expected heterozygosity (H e )

15. Statistical Analysis continued Estimated genetic diversity at 4 time periods: -1891: All individuals from 3 subgroups -1972: SD and WY (Assumed KS extinct) -1985: WY pre-bottleneck (12 indiv.) -1985: WY during bottleneck (7 indiv.)

16. Fitness Used previously published litter sizes Litter size is a measure of female fecundity and kit survival Compared reintroduced SD (1997-2000) and pre-bottleneck WY (1982-1985) # of kits above ground with female; kits were 75-145 days old

17. Results DNA Analysis Amplified DNA from 27 high-quality samples at 24 loci 648 possible genotypes Quality of modern and frozen samples much greater than that of desiccated samples 16 single-locus genotypes dropped from analysis due to poor quality replicate amplifications (did not match initial DNA)

18. Results (con’t) Polymorphism 24 loci polymorphic in black-footed ferrets 21 loci p.m. in at least one species 7 loci p.m. in all three species (3 loci p.m. incl. rarest ferret pops) Allele length in bp (# of alleles) 24 microsattellite markers for three species of Mustela

19. Results (Con’t) No deviation from Hardy-Weinberg Equilibrium was found No significant linkage disequilibrium found No significant difference in allele size among species

20. Results (Con’t) Subpopulations Subpops found to be genetically distinct F st (0.57 +/- 0.15) 16 alleles in 10 loci unique to Great Plains Greater population differentiation between Wyoming and Great plains than between South Dakota and Kansas GP subpops contained many alleles absent in WY, even though sample size was smaller

21. Results (Con’t) Current distribution of populations Small circles- origins of samples Historically, distribution wider (inset) KS and SD more similar than WY

22. Results (Con’t) Relief Map of Wyoming; non-populated regions correlate with mountainous regions

23. Results (Con’t) Significant difference in number of alleles per locus from all ferret species However, no significant difference in black-footed ferret species, specifically

24. Results (Con’t) When did we lose genetic diversity? Tested in four time periods 1891 1972 1985 Pre-Bottleneck During Bottleneck ***Severe loss in diversity between ’72 and ’85– due to extinction of last Great Plains population***

25. Results (Con’t) Fitness stayed relatively the same Litter size did not differ before and after bottleneck Before bottleneck: 115 litters, 4 years = 3.1 kits per litter After bottleneck: 68 litters, 3 years = 3.2 kits per litter

26. Discussion and Conclusion Genetic diversity of black-footed ferrets greatest in undisturbed Kansas grassland Greater He Values More polymorphic loci Higher # alleles/locus Genetic diversity of SD and WY less due to small, isolated nature of habitat Gen. div. positively correlated to pop size

27. Discussion & Conclusion (Con’t) Gen. div. great among subpops Can be only partially explained by genetic drift due to large distance between WY/SD and KS Greatest differentiation between closest subpops (WY/SD) Least differentiation between farthest subpops (SD/KS)

28. Conclusion and Discussion (Con’t) Abolishment of KS population had little effect on genetic diversity Abolishment of SD population, however, substantially reduced heterozygosity, number of alleles, and % polymorphism >>>Core Satellite Model of Population

29. Conclusion and Discussion (Con’t) Small decrease in gen. div. due to bottleneck associated with founder event Establishment of the captive population Litter size did not differ between samples species before, or after the bottleneck

30. Conclusion and Discussion (Con’t) Bottom line: Comparison of genetic diversity among subpops of black-footed ferrets (over 110 years) suggests that genetic diversity was greater prior to the extinction of the Great Plains subpops Bottleneck of WY pop in 1985 induced more loss of genetic diversity Ultimately, population losses, habitat loss, and land conversion account for the loss in genetic diversity