The origin and evolution of ratites, the flightless birds, is widely attributed to the continental drifting of Gondwanaland [1]. One hundred and fifty million years ago, Gondwanaland was a consortium of South America, Arabia, Africa, Australia, Antarctica, India and Madagascar [2]. Initial breakup of this supercontinent during the Early Cretaceous, ~130 to 100 million years ago (Ma), separated Africa and Indo-Madagascar (Fig 1). Later, the remaining Gondwana landmass further split into South America and Australia/Antarctica [1]. During the Late Cretaceous (~80 Ma), Australia/Antarctica and Indo-Madagascar may have been connected through Kerguelen Plateau until India drifted further northwards following its break up from Madagascar [3]. This continental drift theory is complemented by the vicariant speciation that led to the evolution of ratites [1]. In the fractured Gondwana, rhea and emu migrated to South America and Australia, respectively, while elephant birds and ostrich existed together on Indo-Madagascar [2]. Continued northerly movement of India split the Indo-Madagascar landmass at ~88 Ma, with elephant birds remaining in Madagascar and ostriches in India. This biogeographical dispersion eventually led to hopping of ostriches in Africa through Eurasia via a land route around ~20 Ma [3]. The above studies impliy that elephant birds and ostriches should be the oldest ratite lineages. However, recent investigations by Mitchell et al, 2014 suggested that kiwis and elephant birds form the closest group, in contradiction with the continental drift theory [1].

Known occurrences of fossil eggshells of ratites in the Indian subcontinent range in age from the Middle Miocene to Late Pleistocene epochs [4–5]. Aigner reported ratite eggshells in 1935 from the Late Miocene Siwalik rocks of Dhokpathan near Hasnot in Punjab, Pakistan [6]. Evidence from the Siwalik formations revealed the presence of Struthionidae (ostrich family) from the Middle Miocene to Middle Pleistocene. No later records of the family are known from the Himalayan foreland but it is known to have existed in Central India [7]. Skeletal remains of the Siwalik ratites were identified as Struthio asiaticus (1871), whose eggshells differ in size from those of Struthio camelus [8–9]. Recently, AMS dating of eggshells confirmed the earliest evidence of ostriches on the Indian subcontinent and their expansion into Central and Western India, prior to 60 thousand years ago (ka) [5]. The eggshells can be identified on the basis of morphological characters such as eggshell thickness, pore complex shape, density and diameter [10–13]. Eggshells with struthinoid type pore patterns were found in younger Siwalik deposits ranging in age from 2.24 Ma to 0.5 Ma [14]. However, from Indian sites (Rajasthan, Uttar Pradesh, M.P., Maharashtra), fossil eggshells dating between >60000–18000 years ago have been identified as Struthio sp. [6,7,15]. This taxonomic identification is based on the pattern of decreasing pore diameter (1 mm), shell thickness (2.5 cm) and increasing pore density through time in comparison to other ostrich eggshells classified as Struthio karingarbensis, Struthio daberasensis, Struthio kakesiensis and S. camelus from Late Miocene and Pliocene of Namibia and East Africa [5]. Ostrich eggshells with a mean shell thickness of 2.54mm, identified as S. asiaticus, were found in Ahl al Oughlam, Morocco from Late Pliocene deposits. This shell thickness overlaps with the range observed in S. daberasensis, but the latter has a greater pore density comparable to Struthio camelus, thus following the general African trend [16]. The Late Pleistocene ostrich populations in India also show decreased shell thickness and high pore density similar to the African taxa. However, assignment of the Indian material to the African species based on morphological similarity alone may be problematic as this similarity may be a result of similar paleoenvironmental conditions [17–20]. DNA based species identification offers an alternative tool for authenticating the presence of ostriches in Indian peninsula.

Recently, avian fossil eggshells have been characterized as a potent source of preserved aDNA biomolecules [21]. The ultrastructure of eggshells shows a characteristic morphology consisting of several layers (mammillary layer, palisade layer and cuticle layer) beginning from the inner to outer surface (Fig 2). The matrix of the avian eggshell is porous, an ordered and heterogeneous complex, which is coupled with an extracellular structure composed of mineralized and non-mineralized regions having calcium carbonate (CaCO₃) (calcite 97%) and 3.5% organic matrix [18,22–23]. The intracrystalline organic matrix of avian eggshells prevents diffusion losses and isotopic exchange of organic constituents [19,21,24]. This structural hallmark provides an external skeletal support to the developing avian embryo and controls gases, water exchange as well as guards against microbial interventions and physical stresses [21, 25–27]. This structure also imparts stability as well as excellent mechanical strength to the eggshell, besides providing antibacterial and antifouling properties thus enhancing the preservation potential of biomolecules in eggshells [25,28].

In this study, we examined the extent of aDNA preservation in eleven fossilized avian eggshell samples collected from eight archaeological sites in central and western parts of India (S1 Fig). The eggshells used in this investigation have survived harsh environmental conditions [5] and have been dated to Upper Palaeolithic (25,000 to 40,000 years B.P) based on the stone tool technology [6,29–30] and radiocarbon dating at Groningen University Lab, Germany [31,32]. Some of these eggshells are engraved into finished or unfinished beads [33–35]. The perforated beads belong to archaeological assemblages that correspond to the same time period when modern human populations would have dispersed out of Africa [36]. The presence of DNA hotspots in the mammillary layer was detected with DNA binding fluorescent dye Hoechst 33342 and imaged through Confocal Laser Scanning Microscopy. DNA was isolated, quantitized using Bioanalyzer 2100 (Agilent) and a 43 bp sequence of the mitochondrial coding region (16S rRNA) was amplified and sequenced from one of the samples using species-specific primers. The DNA sequence have been submitted to NCBI GenBank (accession number KU251475).

Eleven fossil samples from India, collected from eight archaeological sites (Table 1) and from the personal collections of palaeontologists, were investigated to identify the extent of DNA preservation in these fossil avian eggshells and for DNA based species identification. To maximize the DNA recovery from fossil eggshells, it was important to determine the physical location of DNA and to microscopically identify its presence in the inner, outer or calcified layers of the eggshell.

The breakup of the supercontinent Gondwanaland during the Early Cretaceous resulted in the vicariant speciation of ratites, the flightless birds, to various countries that we know today as South America, Australia, New Zealand, India and Madagascar. The continental drift theory implies that ostriches migrated to India from other landmasses, consistent with the geographical distribution of fossil records of ratites. The fossil eggshells used in this study date to 25–40 ka and morphologically characterized as belonging to Struthio camelus [6,30]. Ostriches were believed to be extinct in India during the Late Pleistocene but our investigation reports the first genetic evidence of their presence on the subcontinent. Fossil eggshells have been reported as being more conducive to biomolecular preservation compared to fossil bones due to their intracrystalline structure, which minimizes microbial contamination [25,28]. This study is significant as it is the first report, to the best of our knowledge, of long-term DNA preservation in fossil eggshells collected from tropical environments with heterogeneous climatic conditions as those encountered in India. aDNA survival in fossils is usually determined by the thermal age theory[46]. This theory requires specific parameters including temperature, pressure and climatic conditions of fossil locations. These parameters are unfortunately not available for our samples, which are from open sites. To evaluate the presence of DNA, Hoechst 33342 stained fossil eggshell samples were visualized under CLSM. DNA hotspots observed on the periphery of mammillary layer are consistent with shape and size of epithelial fossil cells instead of random shapeless patches, eliminating the possibility of these hotspots being of microbial origin. Modifications in the aDNA isolation procedure of Oskam and Bunce [41] were made at the heating step to prevent complete denaturation of aDNA. Species-specific primers based on S. camelus were designed to target short fragments. Amplified fragments were further cloned and sequenced. The multiple alignment of sequences confirms the relationship of our samples with other avian species and their identity with Stuthio camelus. Molecular phylogenetic tree further revealed the placement of our samples in this clade and a basal position relative to other studied paleognaths, which supports the vicariant speciation theory and other ratites phylogenetic studies.

Morphological studies conducted earlier on these eggshells [6,17,30] indicated their close proximity to Struthio species. The present genetic study performed using species-specific primers confirms that the eggshell fragments belong to the Struthio species and that these ratites were present in India during the Late Pleistocene. Paleoenvironmental studies indicate favorable environment for survival and expansion of this species in the Indian subcontinent during Late Pleistocene [5]. Currently, ostriches are extinct in India and their sudden extinction coincides with the period of expansion of humans from Africa towards South Asia [5]. There is evidence in the archaeological context for the occurrence of ostrich eggshell beads and crisscross motif patterns on these eggshells with striking similarities to material culture of Africa [36]. These records suggest a connection between the earliest modern humans in southern Asia and their probable ancestors in eastern and southern Africa [36]. However, no conclusive evidence in this regard is available as yet.

The archaeological records of eggshells fragments, beads and rock shelter paintings discovered at Indian sites are consistent with our genetic study, which is the first molecular evidence indicating the presence of ostriches in India. These evidences further endorse the biogeographical movement of ostriches in India when Indo-Madagascar broke apart [3]. Furthermore, this genetic characterization of fossil eggshells is important in studying aspects related to biology, ecology and extinction of this species.