The first molecular identification of Egyptian Miocene petrified dicot woods (Egyptians’ dream becomes a reality)

Shaimaa S. Sobieh, Botany Department, Faculty of Women for Arts, Science and Education, Ain Shams University, Cairo, Egypt

Mona H. Darwish, Botany Department, Faculty of Women for Arts, Science and Education, Ain Shams University, Cairo, Egypt

Over the past twenty years, several ancient DNA studies have been published, but none has targeted ancient Egyptian DNA. Initial studies on ancient plant DNA were published in the mid-eighties (Golberg et al. 19 91). Rogers and Bendich (1985) reported the extraction of nanogram amounts of DNA from plant tissues ranging in age from 22000 to greater than 44600 years old. DNA from fossils facilitates the calibration of mutation rates among related taxa (Poinar et al. 1993).

Ancient DNA (aDNA) is the most important and informative biological component that scientists can find in archaeological areas for identification purposes. Ancient DNA analysis is used synergistically with other identification methods, such as morphological and anatomical observations and microscopic analyses. DNA barcoding complements the microscopic techniques used in archaeobotany. DNA analysis can be solely used for the identification of specimens when the morphological and anatomical characteristics are absent (Hamalton 2016). Ancient DNA may be used to reconstruct proximal his-tories of species and populations. Studies involving the extraction, sequencing, and verification of fossil DNA demonstrate the existence of material that can be useful to both palaeontologists and evolutionary geneticists.

This opens the possibility for coordinated studies of macro- and microevolutionary patterns that directly approach the relationship between morphological changes on the one hand and genetic changes on the other. In addition, molecular evolutionary studies attempt to reconstruct relationships between concurrent taxa by deducing ancestral states and the genetic distances between them (Golenberg 1994).Ancient wood is found in high abundance, and samples are usually large enough to be analysed. For that reason, wood is an ideal target for ancient plant DNA studies (Kim et al. 2004). However, three problems obstruct the isolation and amplification of DNA from any aDNA specimens (Nasab et al. 2010).

The first is the presence of contamination. The second is the existence of inhibitors of Ta q DNA polymerase in ancient samples, while the third is the small quantity and low quality of DNA that is regained from dead wood (Kaestle and Horsburgh 2002) and this is due to degradation of DNA into small fragments in dead tissue (Deguilloux et al. 2002). Nevertheless, there are several reports of molecular analyses of aDNA from plants. Ancient DNA was extracted from 1600 year-old millet (Panicum miliaceum) by Gyulai et al. (2006)and in 1993, aDNA was extracted from 600- year-old maize cobs (Goloubinoff et al. 1993). Wagner et al. (2018) characterized the aDNA preserved in subfossil (nonpetrified) and archaeological waterlogged wood from the Holocene age (550–9,800 years ago).

DNA barcoding is used to identify unknown samples, in terms of a pre-existing classification (Tripathi et al. 2013) or to assess whether species should be combined or separated. It is also used to establish a shared community resource of DNA sequences that can be used for organismal identification and taxonomic clarification (Tripathi et al. 2013). The nuclear ribosomal internal transcribed spacer (ITS) region is indicated as a plant barcoding region (Hollingsworth et al. 2 011). Miocene fossils are believed to be the best-preserved fossils of Egypt (El-Saadawi et al. 2014).

These fossils are chemically well preserved because of the low oxygen content and cold temperatures of the water in which they were deposited (Kim et al. 2004). DNA sequences can be obtained from Miocene-age plant remains and the success rate is increased through the use of improved methods of DNA extraction and the amplification of small segments of the fossil DNA (Kim et al. 2004).

El-Saadawi et al. (2014) reported that Egypt contains the second largest deposit of Miocene dicot woods in Africa (containing 23 taxa) after Ethiopia that contains 55 taxa. Seven petrified dicot woods were collected from the Wadi Natrun area in Egypt by Prof. Wagih El-Saad-awi and Prof. Marwa Kamal El-Din (Botany Department, Faculty of Science, Ain Shams University). They identified only three of them, namely (Bombacoxylon owenii (Leguminosae/Papilionoideae), Dalbergioxylon dicorynioides (Fabaceae/Faboideae) and Sapindoxylon stromeri (Sapindaceae) based on the wood anatomy (El-Saadawi et al. 2014).

Therefore, the main purpose of the present study was to extract and amplify aDNA from these Egyptian Miocene petrified dicot woods to pro-vide a complete identification. DNA was successfully isolated from the wood samples of Bombacoxylon owenii and Dalbergioxylon dicorynioides. We used molecular techniques to confirm the wood anatomy identification of the two Egyptian wood fossils using DNA barcoding method. In addition, we validated the relationship between the plant fossil woods and the nearest living relative (NLR) based on molecular data acquired from the ITS barcode.


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