Karoo vertebrate dental growth rings

The early diversification of mammalian forerunners (i.e., therapsids) was disrupted by several major biological crises. One of particular interest is the Permian-Triassic mass extinction event which occurred at the end of the Permian (252 Ma) and eradicated about 70% of all terrestrial species 1,2. In this context, the survival strategies employed by therapsids are the subject of major scientific interest. The Karoo Basin in southern Africa provides a particularly well-documented geological and palaeontological record that chronicles the evolution of therapsids throughout this extinction event2. Most significantly, this record shows an increase in fossoriality by land vertebrates3 which co-occurred with an important aridification of the climate between the Late Permian and Early Triassic4. This information led to the hypothesis that therapsids were using burrows and torpor to respectively escape harsh climates and reduce their metabolic rate during prolonged lack of resources5. This burrowing strategy has subsequently been invoked to have played a major role for the survival of mammals during the Cretaceous-Paleogene extinction, 66 Ma, to account for their survival and how they outclassed dinosaurs as the dominant species3. As such, burrowing enabled therapsids and mammals to survive to, and thrive after multiple mass extinctions, making this behaviour the lifeline of mammalian evolution.
However, while burrows do offer thermal refugia in modern ecosystems and modern vertebrates employ torpor to survive droughts6, the proposed hypothesis that therapsids employed torpor and burrowing as survival strategies only relies on circumstantial evidence and is yet to be fully tested. Therefore, investigation of the stated hypothesis entails overcoming long-standing technical challenges. Thus, the objectives are as follows: 1, finding a reliable proxy to measure annual physiological activity and short torpor events in extinct animals; and 2, investigating airflow and thermal mitigation in fossil burrows, precluding direct measurements. Thus, the aim of this proposal is to tackle these two key challenges and assess the role of torpor and burrowing as potential survival strategies in therapsids. For the purposes of this application, the requested specimens are to aid in assessing objective 1.





Objective 1: To assess the life history of therapsids using tooth microstructure.

The primary aim of objective 1 is to determine signs of significant decrease in physiological activity denoting the use of torpor in therapsids. Due to the harsh environmental conditions during the extinction event1,2, it is hypothesised that some animals may have undergone periods of torpor (i.e. a state of dormancy). In doing this, the animal requires far less food and water, allowing it to survive longer periods of drought when resources become scarce7. Periods of torpor would have had short term effects on the animal that may not show in bone8,9, therefore necessitating a smaller unit of measurement. Dentin in the teeth of animals grows in daily incremental layers which accumulate over the animal’s lifespan. Additionally, the continuous tooth replacement pattern in many therapsids allows for analysis of up to three complete tooth replacement cycles in a single tooth row10. Counting daily incremental layers of dentin will allow for the calculation of the duration of a tooth replacement cycle. Furthermore, analysing variations in the incremental growth pattern will inform on the physiological activity of the animal. A preliminary test experiment conducted at the European Synchrotron Radiation Facility (ESRF) has shown that images of the daily increment layers of the dentine can be successfully captured in therapsids. New data will be acquired focusing on well-preserved specimens, thus reducing the impact that weathering and cracks may have on the accuracy of the dentin layer analysis. This project will look at a number of specimens that have been found in burrows, as many animals are presumed to use burrows during periods of torpor7. Thrinaxodon liorhinus11, Diictodon feliceps12, and Microgomphodon oligocynus13 have been selected as the preliminary taxa to be studied as they are represented by exquisitely preserved specimens that should provide excellent data.

Material and methods

The tooth microstructure will be analysed on various therapsid specimens which have been found in burrows (i.e., Thrinaxodon liorhinus11, Diictodon feliceps12, and Microgomphodon oligocynus13), however at this stage of the PhD thesis, the focus will be on specimens of Thrinaxodon. The data set includes the 7 specimens in total (6 specimens that have been requested in this application, and an additional ESI specimen which is already at the ESRF). The Thrinaxodon specimen (BP/7199) contained within the well-known ‘Odd Couple’ burrow (BP/1/5558) was scanned in 201311, with impressive results produced. Due to the recent upgrade of the beamline, the specimen was requested in 2024 so that it can be re-scanned under new parameters for more detailed results. The specimen is currently at the ESRF awaiting scanning, which will be conducted on 25 February 2025. Additionally, it is noted by the application that one of the requested specimens, BP/1/4534, is listed in the ESI Catalogue as a specimen of Trirachodon, however initial CT scanning of BP/1/4534 at the ESI indicates that it may be a juvenile specimen of Thrinaxodon and is therefore included in this study. The ESI CT scan of BP/1/4534 did not provide the type of high-quality resolution of the dentition required for the project, therefore a more detailed scan is required at the ESRF. The skulls will represent several ages (juvenile, sub-adult and adult) based on different skull lengths (the smallest ~37 mm in length, the largest ~90 mm) to ensure sufficient data for comparing tooth replacement cycles to the dentin incremental growth layers in order to accomplish the above-mentioned objectives.

The data for the tooth replacement analysis will be obtained by synchrotron X-ray micro computed-tomography (SR-XCT), which provides detail on the internal cranial anatomy of an animal. Recent improvements in propagation phase contrast synchrotron radiation X-ray micro computed tomography (PPC-SR-XCT) which have occurred at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France indicate that it is now capable of scanning the microstructures present in therapsid fossils, even if the fossil is contained within a burrow cast, to sub-micron level. We propose to perform microtomography at different resolution ranging from 3.5 to 0.7 microns (obtained from an appropriate association of an optic device with the camera). A protocol to use phase contrast by propagation is required to observe the different layer of dentine. The use of filtered pink beam will permit us to have enough flux for image quality and significantly reduce the scanning time per tooth (compared to monochromatic beam). Finally, we will use a PCO.edge camera which will also contribute to reduce the data acquisition time, allowing us to increase our sample set.

To address the objective and scientific questions, several scanning resolutions are needed for each sample. We will favour specimens already scanned at medium resolution (~30 microns) from previous studies11 which already show a general idea of the tooth arrangement (number of teeth, position and location) and replacement status. The assessment of tooth replacement patterns will be achieved by analysing the complete tooth row of each specimen. For all of the specimens, each individual tooth will be scanned with a resolution ranging from 0.7 to 1.4 microns, depending on the size of the considered tooth; large canines might be scanned using an even lower resolution such as 3.5 microns. A resolution about or under 1 micron is indeed crucial to visualize successive layers of dentine resulting from the daily biomineralization process. As Thrinaxodon post-canine teeth are about 2 to 4 mm in maximum width, we could eventually use a half acquisition protocol to extend the lateral field of view. These proposed resolutions will allow the observation of the tooth microstructure, which will then be used to analyse for period of torpor.

Reference list
1. Benton, M. J. & Newell, A. J. Gondwana Res. 25, 1308–1337 (2014).
2. Smith, R. S. et al. in Forerunners of mammals: Radiation, histology, biology. Life of the Past. 31–62 (Bloomington: Indiana University Press, 2011).
3. Marchetti, L. et al. Earth-Sci. Rev. 250, 104702 (2024).
4. Catuneanu, O. et al. J. Afr. Earth Sci. 43, 211–253 (2005).
5. Hildebrand, M. & Goslow, G. E. (John Wiley & Sons, New York, NY, 2001).
6. Fernandez, V. et al. PLoS One 8, e64978 (2013).
7. Geiser, F. & Ruf, T. Physiol. Zool. 68(6), 935-966 (1995).
8. Navas, C. A. & Carvalho, J. E. Aestivation: Molecular and Physiological Aspects. Vol. 49 (Springer Verlag, 2010).
9. Klevezal, G. A. & Kleĭnenberg, S. E. Age determination of mammals from annual layers in teeth and bones. Vol. 69 1-128 (Moscow: Nauka, 1967).
10. Wolvaardt, F.P. Sedimentology and Taphonomy of a tetrapod fossil accumulation in the Triassic Burgersdorp Formation of the Karoo Basin [MSc dissertation, University of the Witwatersrand] (2021).
11. Abdala, F., Jasinoski, S. C. & Fernandez, V. J. Vert. Paleontol. 33, 1408-1431 (2013).
12. Damiani, R. et al. Proc. R Soc. Lond. B, 270, 1747-1751 (2003).
13. Smith, R.M.H. Palaeogeogr. Palaeoclimatol. Palaeoecol. 60, 155–170 (1987).