STUDIES ON ANCIENT DNA AND STABLE ISOTOPE PROFILING OF FOSSILS

Abstract

Ancient DNA (aDNA) analysis of extinct ratite species is of considerable interest as it provides important insights into their origin, evolution, palaeogeographical distribution and vicariant speciation in congruence with continental drift theory. Recent discoveries and methodological advancement in molecular biology, and in particular in sequencing techniques, have rendered successful analysis of ancient DNA (aDNA) from Pleistocene populations and fossil sediments. This genetic information from ancient specimens is invaluable to reconstruct the evolutionary path adopted by species. In contrast to ancient DNA analyses from subfossil bones and teeth, as well as ancient sediments, recovery, and characterization of aDNA from fossil eggshell has been performed recently by Oskam et al. 2010. These eggshells are found frequently in archaeological deposits and are an important source for paleoecology, paleodietary, zooarchaeology and geochronological studies. Moreover, the physical and chemical properties of biomolecules in eggshells were well preserved. Early human settlements and various fossil deposits describe the presence of avian eggshells all over the world. During Pliocene and Pleistocene periods, ratites occupied wide areas of China, Southern Russia, India, Eastern and Southern Europe, Middle East as well as Africa. The present-day distribution of such ratites reveals the continental drift and they are considered to be of Gondwanan origin. Fossil avian eggshells have been found at more than 50 localities in India (Madhya Pradesh, Rajasthan, Maharashtra, and Gujarat). Besides eggshell pieces artifacts such as eggshell beads and bone fragments have also been reported from these sites, which were identified as Struthio asiaticus species. Morphological and ultrastructure characterization of eggshells was done by Scanning Electron Microscopy (SEM), X-Ray Diffraction (XRD), Electron Back Scatter Diffraction (EBSD) as described in Chapter 2. SEM and XRD were conducted at IIC, IIT Roorkee, India, and EBSD was conducted at University of Alabama, USA. Data obtained from biominerals studies was of importance as they provide an understanding of natural evolutionary processes. SEM studies demonstrated the ultrastructure of fossil eggshells and formation of the calcified cuticular layer. The presence of calcified cuticle layer in the eggshell is the basis for ancient DNA studies as it contains preserved biomolecules. EBSD accentuates the crystallographic structure of the ostrich eggshells with sub-micrometer resolution. It is a non-destructive tool for ii evaluating the extent of diagenesis in eggshell. EBSD analysis revealed the presence of dolomite in the eggshells. This research resulted in the complete recognition of the structure of ostrich eggshells as well as the nature and extent of diagenesis in these eggshells which is vital for genetic and paleoenvironmental studies. The primary aim of this research was to extract aDNA from ostrich eggshell and to successfully amplify and sequence it. The sequences obtained were then analyzed phylogenetically and were submitted in NCBI GenBank accession number KU251475 as mentioned in Chapter 3. aDNA extraction was carried out in a dedicated aDNA laboratory at CSIR-CCMB, Hyderabad. DNA hotspots were first observed using Confocal Laser Scanning Microscopy (CLSM). In this chapter, the designing and testing of primers for simplex and multiplex PCR was done that allow analysis of the minute amount of DNA and very short DNA fragments. Optimization of the DNA extraction procedure from eggshells through quantitative real-time PCR was done. Produced sequences were then analyzed in light of phylogeny, biogeography & molecular evolution of the corresponding species. In chapter 4, Stable isotopic profiles of eggshells have established that help in the study of paleobiology and paleodietary. Carbon and oxygen stable isotope were analyzed for the diets and habitat of the ostriches. Carbonate eggshells were analyzed using acid digestion method. Released carbon dioxide was passed through Gas Chromatography and then analyzed by IMRS (Isotope Mass Ratio spectrometer). Carbon isotope revealed the diets of the ostrich to be a mixture of C3 and C4 plants with biases toward C3 shedding the light on the paleoecology and paleodietary. Oxygen isotopes indicate the aridity and less humid conditions. In the next chapter, triple oxygen isotopes techniques have been detailed. Isotope analysis was conducted at stable isotope lab of Johns Hopkins University. Body water model for the animal has been developed to study the palaeoenvironment aspects. Eggshells possess the biochemical signature of the ostrich. The sample was digested with acid digestion and carbon dioxide was converted into water and then into oxygen to calculate the 17 mass isotope of oxygen. This isotope is in very low abundance and thus its calculation with maximum precision and minimum error is a tough task. State of art mass spectrometry was used to analyze this isotope. D17O values of samples lie below the maximum evaporation model suggesting that relative humidity and water economy index to low. It indicates that ostriches are from arid climates and consumed lots of evaporated leaf waters. The research revealed how these samples can provide insights into the biology, ecology and extinction of this species. Last chapter summarizes the main findings and the importance of the results and also leads path for future directions.