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

Introduction Crocodylus niloticus

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

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

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

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

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

Materials and Methods 8

Sampling Sites 9

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

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

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

Results and Discussion - mtDNA Origin of C. niloticus 13

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.

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* 0.07275* 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

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

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 (17.7-432.7) 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 (74.5-743.8) Lower Shire River (South) 43.4 (29.4-75.2)

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

Acknowledgement 19

References Schmitz, A., Mansfeld, P., Hekkala, E., Shine, T., Nickel, H., Amato, G. and Bohme, W., 2003. Molecular evidence for species level divergence in African Nile crocodile Crocodylus niloticus (Laurenti, 1786). Science Direct 2:703-712. Bishop, J. M., Leslie, A. J., Bourquin, S. L. and O’Ryan, C., 2009. Reduced effective population size in an overexploited population of the Nile crocodile (Crocodylus niloticus), Biological Conservation 142:2335-2341. [Image 1] http://www.vecteezy.com/map-vector/5908-map-vector-of-africa [Image 2] http://www.iucnredlist.org/details/46590/0 [Image 3] http://photography.nationalgeographic.com/photography/photo-of-the-day/mother-child-botswana-pod/ [Image 4] http://www.businessoffashion.com/articles/news-analysis/crocodile-bites-show-why-your-birkin-bag-is-so-expensive [Image 5] https://www.change.org/p/alberta-development-partners-llc-recreate-the-prairie-ecosystem-with-appropriate-prairie-dog-habitat-and-an-on-site-educational-program-to-atone-for-the-merciless-slaughter-of-1000-prairie-dogs-in-castle-rock-colorado