Does sperm biology inform us about the phylogenetic relationships among elephant, manatee and hyrax? Gerhard van der Horst1, Danielle Arnold2, Jonathan Cowart2, Iske Larkin2, Chris Mortensen2 1University of the Western Cape, South Africa, 2University of Florida, USA gvdhorst7@gmail.com INTRODUCTION There has been major controversy in terms of the phylogenetic relationships in Afrotheria and particularly which is closer related to the elephant, the manatee or the hyrax? Older/newer literature considers the hyrax a closer ally but recent information indicates that the elephant might have emerged secondarily from the water and accordingly share more features with manatee. We ask if sperm of these species show some co-evolution in terms of their evolutionary history and to what extent do they show relationships in terms of aspects such as sperm competition. We used electron microscopy, and computer aided semen analysis to quantitatively analyze, sperm motility, morphometrics and vitality to help us evaluate these relationships. Species Length µm Width µm Perimeter µm Area µ2 Hyrax 5.3 3.6 12.83 18.76 Elephant 6.9 3.35 15 21 Manatee 7.7 3.37 16.4 26.3 Table 1: Sperm Morphometric parameters among the three species Fig. 2: Transmission electron microscopy showing similar features of the midpiece, Left: Hyrax; Middle: African elephant; Right:Manatee MATERIALS AND METHODS Semen was collected from hyrax (Procavia capensis)(A), African elephant (Loxodonta africana)(B), and Florida manatee, (Trichechus manatus latirostris)(C) Motile sperm was selected via swim-out/flush methods in Ham’s F10. Sperm morphometric parameters were determined after SpermBlue staining and analysis via Sperm Class Analyzer (SCA), and transmission electron microscopy (TEM). Sperm motility and sperm morphology was determined by means of computer aided sperm motility analysis (CASA) using the SCA system. Statistical analyses were performed using MedCalc . Species Motil % VCL µm/s VSL µm/s LIN % Hyrax 80 135 110 78 Elephant 290 255 89 Manatee - Table 2: selective CASA sperm motility features of hyrax and elephant. Sperm in African elephant swim significantly faster than in hyrax and also more linearly as can be seen in both VSL and LIN. Manatee sampling will take place in the next few months to complete the motility similarities/ differences. A B C DISCUSSION AND CONCLUSION There are numerous similarities among hyrax, African elephant and manatee in terms of many anatomical features including the anatomy and histology of the reproductive system. Based on these aspects alone it is difficult to establish best phylogenetic affinity. Our study investigated the hypothesis that sperm characteristics may assist to solve these relationships. Our preliminary findings suggest that on the basis of sperm size, sperm morphometric parameters and potentially sperm motility kinetics, African elephant and manatee are closer related than to hyrax. On the basis of the fused pleurae, African elephant might have secondarily gone back to a water existence before finally coming to land. In this process there might have been a greater degree of co-evolution among AE and M than H. ACKNOWLEDGEMENTS The support of the three institutions below as well as using in some instances the data on AE of Luther (2017): PhD thesis, UWC RESULTS Fig. 1 shows the light microscopic features of the sperm of the three species and Table 1 depicts the morphometric data for the three species. The sperm head of hyrax (H) is considerably and significantly smaller in length and perimeter than that of African elephant (AE) and manatee (M) while the latter two species share similar morphometric characteristics. Some of these similarities are also seen in TEM micrographs. In hyrax, there is extensive acrosome covering (two thirds of the head). In contrast in both AE and M the acrosome is large but not as extensive as in H. There seems to be 35 to 40 mitochondrial gyres relating to about 70 to 80 mitochondria in all three species (Fig. 2). Fig.1: Morphology of Hyrax (left) African elephant (middle) and Manatee (right) (same scale). Hyrax notably smaller than AE and M 14th Biology of Sperm Sheffield 2017