1. Track the Split of Crocodile Sub Populations
Versfeld WF, Leslie AJ, van Asch B, Mathews I, Beytell PC, Du Preez P, Rhode C, Slabbert R (2016) 1

2. Introduction
Crocodylus niloticus

3. Introduction - mtDNA Mother to off spring Non-recombinant
Mutates very slowly
Mutations passed down from mother to off spring
Deciphering genealogical history
-Power House of the cell
-Passed down from the mother to her off spring
Non-recombinant (does not combine with other DNA)
Mutates very slowly
If a mutations occurs it is passed down from mother to off spring
mtDNA sequences are used as a tool for deciphering genealogical history

4. Introduction - mtDNA - Schmitz et al. 2003
Origin of C. niloticus
Hekkala et al. 2011
- Schmitz et al. 2003
- Crocodile is a diverse species
- Two independent clades
West & East Africa
- Result of Geographical
separation
- Is there two species of Nile
crocodile in Africa?
Origin of C. suchus

5. Introduction - STRs Short Tandem repeats or microsatellites
Highly variable DNA regions
Co-dominant markers
Polymorphic loci
Non-coding region of the DNA
Commonly used for:
population diversity;
population structure;
fingerprinting;
Pedigree inference etc.
Non-coding
Cheap to use and cross amplifiable between common species
No need to develop them as that is costly

6. Introduction Eastern or western Nile crocodile populations
Where did they come from?
Where they previously connected or not? 6

7. Aim
Genetic diversity of the Namibian Nile crocodile in the Kunene & Okavango River systems
Evaluate structure of Southern African Nile crocodile populations by the use of molecular tools (mtDNA and STR loci)
Hypothesis
The two populations in the river systems diverged due to geographical or other physical barriers to form distinct genetic populations with no recent gene flow expected 7

8. Materials and Methods 8

9. Sampling Sites 9

10. Methodology - mtDNA EXTRACT DNA AMPLIFY mtDNA MARKERS SEQUENCE
GENETIC DATA ANALYSES 10

11. Methodology - STRs EXTRACT DNA CROSS-SPECIES MULTIPLEX DESIGN GENOTYPE
GENETIC DATA ANALYSES 11

12. STR Methodology Hardy Weinberg Equilibrium Null Hypothesis
Population structure
Sub-Population
Global
Diversity
Meta-Population
Sub-Populations
He + Ho = Fis Na
Rs or Ae
Extinction Risks
Effective Population Size
Bottlenecks
Structure
-Pairwise Fst
AMOVA = % variation and Global Fst
Structure Bayesian Clusters

13. Results and Discussion - mtDNA
Origin of C. niloticus 13

14. Results and Discussion - STRs
Population N An He Ho
HWE
(P-value) Rs
Fis
Panmixia
139 11
0.71
0.61
0.003*
10.85
0.144
Lower Kunene River 12
4.2
0.58
0.5
0.587
4.097
0.149
Bwabwatwa National Park 20
5.3
0.6
0.616
4.481
-0.01
Okavango Delta 29 3
0.88
0.75
0.613
4.749
0.15
Otjiwarongo Crocodile Farm 13
4.3
0.56
0.54
0.537
4.137
0.043
Okavango 62
5.2
0.531
4.456
0.015
Lower Shire River (North) 27
6.9
0.66
0.62
0.337
5.519
0.098
Lower Shire River (South) 25
0.68
0.63
0.367
5.54
0.071
Shire 52
0.67
0.352
5.529
0.085
* = Values that indicate significant differentiation, P<0.05.

15. Results and Discussion - STRs
Lower Kunene River
Bwabwatwa National Park
Okavango Delta
Otjiwarongo Crocodile Farm
Lower Shire River (North)
Lower Shire River (South) -
-0.012
-0.034
0.075
0.902*
0.977*
0.138*
-0.046
-0.044
0.896*
0.967*
0.116*
0.005
0.034
0.920*
0.975*
0.055* *
0.882*
0.955*
0.222*
0.175*
0.171*
0.174*
0.008
0.200*
0.159*
0.158*
0.160*
0.003
* = Values that indicate significant differentiation, P<0.05

16. Results and Discussion - STRs
1.00
0.80
0.60
0.40
0.20
0.00 1 2 3 4
Okavango
Lower
Kunene
South Africa Comm.
Lower Shire
1.00
0.80
0.60
0.40
0.20
0.00 1 2
Okavango
Lower Kunene

17. Results and Discussion - STRs
Populations
LD test for Ne
Heterozygosity excess test for Ne
[95% CI]
Lower Kunene River
∞ (24.7-∞)
∞ (∞-∞)
Bwawatwa National Park
38.6 ( )
404.6 (107.3-∞)
56.2 (10.1-∞)
∞ (16.7-∞)
Okavango Delta
292.3 (44.7-∞)
Otjiwarongo Crocodile Farm
62.04(18.1-∞)
Lower Shire River (North)
754.4 (54.1-∞)
143.5 ( )
Lower Shire River (South)
43.4 ( )

18. Conclusion Namibian Nile crocodiles belong to the Eastern Clade;
Lower Kunene and Okavango river systems did not show divergence, no mtDNA mutational differences, however STR analyses showed that the Okavango sample was sub structured;
Comparison of the Lower Kunene and Okavango samples to South Africa and Lower Shire indicated two separate populations;
The genetic diversity of Nile crocodile populations within the Lower Kunene and Okavango River systems was comparable to previously published neighboring river systems;
Based on our findings, the Lower Kunene and Okavango are isolated populations
Contribute to genetic knowledge of freshwater species in Southern Africa,
conservation plan in Namibia 18

19. Acknowledgement 19

20. References
Schmitz, A., Mansfeld, P., Hekkala, E., Shine, T., Nickel, H., Amato, G. and Bohme, W., Molecular evidence for species level divergence in African Nile crocodile Crocodylus niloticus (Laurenti, 1786). Science Direct 2:
Bishop, J. M., Leslie, A. J., Bourquin, S. L. and O’Ryan, C., Reduced effective population size in an overexploited population of the Nile crocodile (Crocodylus niloticus), Biological Conservation 142:
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