The distribution of genetic diversity in sleepy lizards

Author: Talat Hojat Ansari Komachali

Hojat Ansari Komachali, Talat, 2015 The distribution of genetic diversity in sleepy lizards, Flinders University, School of Biological Sciences

Terms of Use: This electronic version is (or will be) made publicly available by Flinders University in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. You may use this material for uses permitted under the Copyright Act 1968. If you are the owner of any included third party copyright material and/or you believe that any material has been made available without permission of the copyright owner please contact with the details.



Understanding the distribution of genetic diversity in and among organisms provides a cornerstone for asking fundamental questions in evolutionary biology. These questions are advanced by connecting the processes affecting genetic diversity within and among individuals, populations and species. Already a strong discipline, molecular ecology has been undergoing a recent resurgence due to dramatic advances in the tools of the trade, the development and availability of molecular markers accessing both neutral and functional areas of genomes. Thanks to these approaches, molecular ecologists have increasing abilities to assess the patterns of genetic variation within and among different levels of life – individuals, populations and species. Multiple markers are necessary to provide a more unbiased view of the many processes and the potential factors that might have contributed to the distribution of current genetic diversity.

Here, I characterise and apply a variety of molecular markers in a widespread monogamous Australian skink Tiliqua rugosa. This species is a member of the Egernia group of skinks which contains species with many varied social structures. My thesis consists of several marker development chapters for both neutral and functional markers; a chapter on the development of tools for obtaining data from next generation sequencing runs; and culminates in a chapter examining the distribution of current genetic diversity and the inference of the historical factors that have led to the observed pattern. First, I used next generation shotgun sequencing of T. rugosa genomic DNA to develop primers for 48 anonymous nuclear loci (ANL).These primers were extensively tested in T. rugosa, and the related Tiliqua adelaidensis and Egernia stokesii. These polymorphic loci provide a useful source of neutral variation for the study of phylogeography and population genetic structure explored later in my thesis. The next two chapters describe the use of transcriptome sequencing for the characterization of markers for a highly diverse gene region which is involved in the immune response and mate choice, the Major Histocompatability Complex (MHC). I designed specific primers from the resultant sequence assembly and successfully amplified exon 2, 3 and 4 from MHC class I, and exon 2 and 3 in class II. The loci were confirmed to be Mendelianly inherited via the sequencing of several families and the utility of these primers was further tested for the related T. adelaidensis and E. stokesii. The characterization of these markers is rare in squamates and represents a breakthrough for investigation in the Egernia group of social lizards and should provide the basis for much further study.

In the next chapter I used these loci and adapted a newly developed technique to provide a cost effective method to amplify the multiple loci from a large number of individuals. I then used these amplifications, of both the neutral and functional loci from 250 T. rugosa individuals, to test bioinformatics software (MITAGSORTER) that I developed to sort the large complicated data sets I obtained from next generation sequencing.

Finally, I used the distribution of genetic variation in samples of T. rugosa from across the entire distribution range of the species in Australia to assess historical processes that potentially were responsible for the contemporary pattern of genetic structure in T. rugosa. The pattern of genetic diversity was quantified using mitochondrial (sequences from ND4) and nuclear (sequences from non-encoding intron 7 of β-fibrinogen, glyceraldehyde-3-phosphate dehydrogenase and nine ANL I developed) markers. This study revealed three major mitochondrial lineages for T. rugosa, although potential secondary admixture was detected, based on nuclear markers. In order to further investigate the potential historical events associated with these phylogeographic patterns, I combined species distribution modelling and a phylogeographic diffusion model. The analyses suggest that the range of T. rugosa contracted during the Last Glacial Maxima (LGM) and that the region around the Murray River and the Nullarbor Plain acted as barriers to gene flow since this period. However there is the possibility that other factors, such as selection on mtDNA, may also have contributed to the pattern of variation observed. My study hints at the presence of multiple refugia for this species during previous glacial maxima, with potential secondary contact following climate amelioration.

My thesis provides valuable resources for the further investigation of the distribution of genetic diversity within this iconic lizard species at scales spanning from individual interactions and mate choice through to a thorough investigation of the role of various ecological processes on the patterns of contemporary genetic diversity. In addition the markers developed can be utilized in investigations at similar scales for many other species within this group of lizards, the Egernia group, with their unusual and varied social systems.

Keywords: Genetic diversity, Phylogeography, Mitochondrial marker, Nuclear marker, Next generation sequencing, Anonymous nuclear loci, Major histocompatibility complex

Subject: Biological Sciences thesis

Thesis type: Doctor of Philosophy
Completed: 2015
School: School of Biological Sciences
Supervisor: Dr. Michael G. Gardner