Sex and stress: Is stress both a mediator and a consequence of sex reversal in the central bearded dragon (Pogona vitticeps)? by Meghan A. Castelli BSc (Hons) A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) July 2021 Institute for Applied Ecology University of Canberra, Australia i ii Abstract Abstract Among vertebrates, sex determining systems are diverse and range on a continuum from entirely genetic sex determination (GSD) to purely environmental sex determination (ESD). The central bearded dragon (Pogona vitticeps) possesses a heterogametic (ZZ male/ZW female) system of genetic sex determination, but high egg incubation temperatures induce sex reversal, in which ZZ genotypic males develop as female. The biological mechanism by which temperature is translated into a sexual outcome is not fully understood in reptiles but is proposed to involve the vertebrate stress axis, a highly conserved environmental sensory mechanism which generates physiological responses to stress through glucocorticoid hormone production. Here I demonstrate using developmental transcriptomes and chemical manipulation experiments that the stress axis is unlikely to mediate sex reversal, and instead find evidence for the involvement of oxidative stress responses or circadian rhythm regulation in high temperature sex reversal. The relevance of these molecular studies is contextualised by a range-wide study of sex reversal and population genetic structure which demonstrates the lack of a clear relationship between climate and sex reversal in the wild. I have proposed that the threshold temperature for sex reversal (and thus the underlying genetic network which determines the threshold) varies across the landscape, having evolved in response to higher average incubation temperatures in warmer regions of the species range. Together, these studies demonstrate that not only is temperature sex reversal in reptiles a molecular process likely driven by sensory mechanisms other than the stress axis, but it also has complex evolutionary dynamics in the wild. iii iv Authorship of Thesis Authorship of Thesis Except where clearly acknowledged in footnotes, quotations and the bibliography, I certify that I am the sole author of the thesis submitted today entitled: Sex and stress: Is stress both a mediator and a consequence of sex reversal in the central bearded dragon (Pogona vitticeps)? I further certify that to the best of my knowledge the thesis contains no material previously published or written by another person except where due reference is made in the text of the thesis. The material in the thesis has not been the basis of an award of any other degree or diploma except where due reference is made in the text of the thesis. The thesis complies with University requirements for a thesis as set out in the Examination of Higher Degree by Research Theses Policy: https://www.canberra.edu.au/research/current- research-students/hdr-policy-and-procedures 05/02/2021 Candidate’s Signature Date Arthur Georges. 05/02/2021 Primary Supervisor’s Signature Date v vi Publications Publications The following chapters have been peer reviewed and published in scientific journals. Chapter 2: Cellular calcium and redox regulation: The mediator of vertebrate environmental sex determination? Castelli, M. A., Whiteley, S. L., Georges, A., & Holleley, C. E. (2020). Cellular calcium and redox regulation: the mediator of vertebrate environmental sex determination? Biological Reviews, 95(3), 680–695. https://doi.org/10.1111/brv.12582 Chapter 4: Corticosterone does not have a role in temperature sex reversal in the central bearded dragon (Pogona vitticeps) Castelli, M., Georges, A., & Holleley, C. E. (2021). Corticosterone does not have a role in temperature sex reversal in the central bearded dragon (Pogona vitticeps). Journal of Experimental Zoology Part A: Ecological and Integrative Physiology, Early view. https://doi.org/10.1002/jez.2441 Chapter 6: Evolving thermal thresholds explain the distribution of temperature sex reversal in an Australian dragon lizard Castelli, M. A., Georges, A., Cherryh, C., Rosauer, D. F., Sarre, S. D., Contador-Kelsall, I., & Holleley, C. E. (2020). Evolving thermal thresholds explain the distribution of temperature sex reversal in an Australian dragon lizard. Diversity and Distributions, Early view. https://doi.org/10.1111/ddi.13203 vii viii Declaration of Co-Authored Publications Declaration of Co-Authored Publications Declaration for Thesis Chapter 2: Cellular calcium and redox regulation: The mediator of vertebrate environmental sex determination? DECLARATION BY CANDIDATE In the case of Chapter 2, the nature and extent of my contribution to the work was the following: Nature of Contribution Extent of Contributions (%) Synthesis of idea, literature review and 50% writing The following co-authors contributed to the work: Name Nature of Contribution Contributor is also a UC student (Yes/No) Sarah L. Whitelely Co-first author. Equal party to Yes synthesis and writing. Arthur Georges Contribution did not exceed that No normally expected of a supervisor, covering all aspects of the work. Clare E. Holleley Contribution did not exceed that No normally expected of a supervisor, covering all aspects of the work. ______________________________________ 20/01/2021 Candidate’s Signature Date DECLARATION BY CO-AUTHORS The undersigned hereby certify that: (1) the above declaration correctly reflects the nature and extent of the candidate’s contribution to this work, and the nature of the contribution of each of the co-authors. (2) they meet the criteria for authorship in that they have participated in the conception, execution, or interpretation, of at least that part of the publication in their field of expertise; ix Declaration of Co-Authored Publications (3) they take public responsibility for their part of the publication, except for the responsible author who accepts overall responsibility for the publication; (4) there are no other authors of the publication according to these criteria; (5) potential conflicts of interest have been disclosed to (a) granting bodies, (b) the editor or publisher of journals or other publications, and (c) the head of the responsible academic unit; and (6) the original data are stored at the following location(s) and will be held for at least five years from the date indicated below: [Please note that the location(s) must be institutional in nature, and should be indicated here as a department, centre or institute, with specific campus identification where relevant.] Location(s): University of Canberra, Institute for Applied Ecology Signatures Date Arthur Georges. 20/1/21 20/1/21 20/1/21 x Declaration of Co-Authored Publications Declaration for Thesis Chapter 4: Corticosterone does not have a role in temperature sex reversal in the central bearded dragon (Pogona vitticeps) DECLARATION BY CANDIDATE In the case of Chapter 4, the nature and extent of my contribution to the work was the following: Nature of Contribution Extent of Contributions (%) Experimental design, data collection, 80% analysis and writing The following co-authors contributed to the work: Name Nature of Contribution Contributor is also a UC student (Yes/No) Arthur Georges Contribution did not exceed No that normally expected of a supervisor, covering all aspects of the work. Clare E. Holleley Contribution did not exceed No that normally expected of a supervisor, covering all aspects of the work. ______________________________________ 20/01/2021 Candidate’s Signature Date DECLARATION BY CO-AUTHORS The undersigned hereby certify that: (7) the above declaration correctly reflects the nature and extent of the candidate’s contribution to this work, and the nature of the contribution of each of the co-authors. (8) they meet the criteria for authorship in that they have participated in the conception, execution, or interpretation, of at least that part of the publication in their field of expertise; (9) they take public responsibility for their part of the publication, except for the responsible author who accepts overall responsibility for the publication; (10) there are no other authors of the publication according to these criteria; xi Declaration of Co-Authored Publications (11) potential conflicts of interest have been disclosed to (a) granting bodies, (b) the editor or publisher of journals or other publications, and (c) the head of the responsible academic unit; and (12) the original data are stored at the following location(s) and will be held for at least five years from the date indicated below: [Please note that the location(s) must be institutional in nature, and should be indicated here as a department, centre or institute, with specific campus identification where relevant.] Location(s): University of Canberra, Institute for Applied Ecology Signatures Date Arthur Georges. 20/1/21 20/1/21 xii Declaration of Co-Authored Publications Declaration for Thesis Chapter 5: Evolving thermal thresholds explain the distribution of temperature sex reversal in an Australian dragon lizard DECLARATION BY CANDIDATE In the case of Chapter 5, the nature and extent of my contribution to the work was the following: Nature of Contribution Extent of Contributions (%) Data collection, analysis, and led 80% manuscript writing The following co-authors contributed to the work: Name Nature of Contribution Contributor is also a UC student (Yes/No) Caitlin Cherryh Code development and data No analysis in collaboration with the candidate