1. Species identification (taxonomy) Population/stock differentiation Individual identification Uses of population genetic data

2. Genetics questions relevant to conservation biology: are x and y different species? are populations genetically different? how much variation is present in a population? how much variation has been lost? which parents contributed to the breeding population? how much migration is occurring between populations?

3. A T T A G C C G T A A T DNA sequence DNA strand chromosome protein organism

4. morphometric and meristic counts ~ 40 characters Often lethal

6. Morphometrics (measurements) 6-7 pelvic fin rays meristics (counts)

7. Issues with morphometric and meristic data Are they different enough? What is the significance of the differences? (do they represent genetic differences?) Do the differences indicate reproductive isolation?

8. protein loci ~ 200 loci Often lethal morphometric and meristic counts ~ 40 characters Often lethal LEVELS OF VARIATION

9. Protein electrophoresis Basis: alleles result from changes in base pairs in DNA code base pair change results in different amino acid in protein resulting proteins may vary in size, shape, net electric charge + + + + + + + + + +

10. Protein electrophoresis Basis: electrophoresis separates proteins on the basis of their net electric charge, and size/shape thus, alleles coding for proteins with different size or charge will be detected as different genotype is deduced from protein types co-dominant, low variability

11. Population 1 a’ a alleles 1 2 3 4 5 6 7 8 9 10 Protein electrophoresis INDIVIDUALS + _

12. Population 1 Population 3 Population 2 a’’ a’ a a’’ a’ a a’’ a’ a alleles 1 2 3 4 5 6 7 8 9 10 Protein electrophoresis INDIVIDUALS

13. monomeric protein dimeric protein

14. protein loci ~ 200 loci Often lethal morphometric and meristic counts ~ 40 characters Often lethal LEVELS OF VARIATION chromosome 100s of characters May be lethal

15. Cytogenetics – information from chromosome number, shape, banding patterns

16. DNA strand 100s to 1,000s of characters Non-lethal protein loci ~ 200 loci Often lethal morphometric and meristic counts ~ 40 characters Often lethal LEVELS OF VARIATION chromosome 100s of characters May be lethal

17. DNA Mitochondrial circular small - 16,000-20,000 bp many copies maternal inheritance moderate variation mostly coded loci Nuclear linear 3 billion + base pairs two copies per cell bi-parental inheritance high variation much non-coded DNA

18. ‘Problems’ with DNA not very much of it (2-100 copies per cell) - need to ‘amplify’ it too much of it (3 billion base pairs) - need to look at small pieces at a time different areas of DNA have different variation

19. Amplification of DNA PCR - polymerase chain reaction: –split DNA strand into two strands –bind primers on either side of segment to be amplified –allow new matching DNA strands to assemble on each side –repeat as often as needed

20. Requires ingredients: DNA template DNA polymerase free nucleotide bases DNA template must be separated (denatured, unwound) must be a primer for DNA polymerase to add free nucleotides to Polymerase Chain Reaction (PCR) - DNA replication in a test tube

21. Polymerase Chain Reaction (PCR) each replication cycle doubles amount of DNA

22. Requires ingredients: DNA template DNA polymerase free nucleotide bases DNA template must be separated (denatured, unwound) heat will denature DNA, but deactivates protein enzymes use Taq DNA polymerase (from bacterium Thermus aquaticus from thermal vents) Polymerase Chain Reaction (PCR)

24. Requires ingredients: DNA template DNA polymerase free nucleotide bases DNA template must be separated (denatured, unwound) must be a primer for DNA polymerase to add free nucleotides to short sequence of DNA that binds ‘upstream’ of area to be replicated Polymerase Chain Reaction (PCR)

25. DNA analyses mtDNA (mitochondrial DNA analysis) RFLP (restriction fragment length polymorphism) RAPD (randomly amplified polymorphic DNA) AFLP (amplified fragment length polymorphism) microsatellites (SSR, simple sequence repeats) single nucleotide polymorphisms (SNPs)

26. A T T G A C T T A A G C G T A G T A A C T G A A T T C G C A T C cleavage site (palindrome) Restriction Fragment Length Polymorphism (RFLP) - cut DNA with restriction enzymes - isolate cut fragments based on length (electrophoresis) - deduce length of fragments - individuals differ based on mutations at restriction sites

27. Restriction enzyme cleavage of mitochondrial DNA Mitochondrial DNA DNA fragments gel

28. A T T G A C T T A A G C G T A G T A A C T G A A T T C G C A T C A T T G A C T C A A G C G T A G T A A C T G A G T T C G C A T C Single base pair substitution removes cleavage site recognition

29. Microsatellite DNA tandem repeats of short DNA sequences (e.g. ACACACACAC…) number of repeats is highly variable – easy cross-over ‘mistake” co-dominant

30. Microsatellite DNA tandem repeats of short DNA sequences (e.g. ACACACACAC…) number of repeats is highly variable – easy cross-over ‘mistake” co-dominant isolate portions of DNA with primers (time-consuming to develop) separate fragments by length ~ number of repeats

31. SNPs (single nucleotide polymorphisms) DNA sequencing Genome sequencing Barcode of Life Database (BOLD)

32. DNA strand 100s to 1,000s of characters Non-lethal protein loci ~ 200 loci May be lethal morphometric and meristic counts ~ 40 characters Often lethal LEVELS OF VARIATION chromosome 100s of characters May be lethal A T T A G C C G T A A T DNA sequence millions… Non-lethal whole frozen live preserved or dried organism tissue tissue tissue

33. Step 1: find markers ‘survey’ species/population for polymorphisms before conducting full study = # of loci, # alleles/locus Step 1(b): find/develop primers for PCR often available on web databases from related taxa Step 2: determine how many markers are needed depends on question(s) of interest

34. MDH-1 MPI Main lake Mallets Bay Inland Sea Smelt isozymes, Lake Champlain

35. XDH-1 LDH-1 Perca flavescens Perca fluviatilis Comparison of Eurasian and N. American yellow perch

36. O’Brien et al. 1983. The cheetah is depauperate in genetic variation - using protein electrophoresis - assumed to be result of small N, bottleneck, then inbreeding - highly vulnerable to disease outbreaks (50% mortality in one captive population) Cheetah ( Acinonyx jubatus):

37. O’Brien et al. 1983. The cheetah is depauperate in genetic variation - using protein electrophoresis ##% poly.av. Speciespopns. NlocilociH Drosophila43 >1002443.10.14 Mus2 874620.50.088 Homo sapiensmany>10010431.70.063 Felis catus1 5655220.076 Cheetah2 55470.00.0

38. Cheetah ( Acinonyx jubatus): Menotti-Raymond and O’Brien et al. 1993. - using protein electrophoresis, high-resolution PE, and mtDNA ##% poly.av. Speciespopns. NlocilociH Drosophila43 >1002443.10.14 Mus2 874620.50.088 Homo sapiensmany>10010431.70.063 Felis catus1 5655220.076 Cheetah2 55470.00.0 Drosophila1 205411.10.04 Mus1 72 4.10.02 Homo sapiens1 34many 1.20.063 Cheetah1 6155 3.20.013 Felis catus1 1746.0 Cheetah3 7645.0

39. Merola, 1994. A reassessment of homozygosity …. - carnivores tend to show low levels of genetic variation (several have lower levels of H and P than cheetah) - measures of fluctuating asymmetry indicate cheetah is not suffering from low homozygosity or genetic stress - sperm deformities – do not affect fertility, may be normal in felids - low litter sizes – in captivity (high in wild) - susceptibility to disease – may be due to captive contact (in wild, cheetahs avoid conspecifics) Concluded that conservation is better directed at habitat

40. Spotted owls vs. barred owls (Haig et al. 2004, Cons. Biol. 18:1347-1357) Northern spotted owl – endangered Barred owl – rapid range expansion has led to overlap with spotted owl - potential competition - potential for hybridization mtDNA and AFLP analysis: - species are distinct - no evidence of previous gene flow - hybrids occur with male spotted and female barred owls - hybrids can be identified until F2 generation

41. e-DNA (environmental DNA) as a method for detection of rare/elusive species