Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 73(2): 3-13, 2020 Firenze University Press www.fupress.com/caryologiaCaryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-750 Citation: S.S. Sobieh, M.H. Darwish (2020) The first molecular identifica- tion of Egyptian Miocene petrified dicot woods (Egyptians’ dream becomes a reality). Caryologia 73(2): 3-13. doi: 10.13128/caryologia-750 Received: December 1, 2019 Accepted: March 13, 2020 Published: July 31, 2020 Copyright: © 2020 S.S. Sobieh, M.H. Darwish. This is an open access, peer-reviewed article published by Firenze University Press (http://www. fupress.com/caryologia) and distributed under the terms of the Creative Com- mons Attribution License, which per- mits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. The first molecular identification of Egyptian Miocene petrified dicot woods (Egyptians’ dream becomes a reality) Shaimaa S. Sobieh*, Mona H. Darwish Botany Department, Faculty of Women for Arts, Science and Education, Ain Shams Uni- versity, Cairo, Egypt E-mail: shimaa.sobieh@women.asu.edu.eg; mona.darwish@women.asu.edu.eg *Corresponding author Abstract. This is the first work on Egyptian ancient DNA (aDNA) from plant fossil remains. Two aDNA extracts from Miocene petrified dicot woods were successful- ly obtained, amplified, sequenced and recorded for the first time in the world using a DNA barcoding technique. Internal transcribed spacers (ITS) barcoding is a tech- nique for delimiting and identifying specimens using standardized DNA regions. The two Miocene dicot woods: Bombacoxylon owenii (Malvaceae/Bombacoideae) and Dalbergioxylon dicorynioides (Leguminosae/Papilionoideae) were collected from the Wadi Natrun area in Egypt and were identified by palaeobotanists on the basis of wood anatomy. The molecular identification by ITS region of Bombacoxylon owe- nii did not match the wood taxonomic assignation. The molecular identification of Bombacoxylon owenii suggested that it is more related to the extant genus Ceiba rather than to the extant genus Bombax. In contrast, the molecular identification by ITS of Dalbergioxylon dicorynioides matched the identification of the palaeobotanist (related to extant genus Dalbergia). Therefore, we suggest that this region should be used as a starting point to identify several plant fossil remains and this work will be helpful in solving problems related to the identification of plant fossils. Keywords: Egyptian petrified woods, aDNA, DNA barcoding, ITS. INTRODUCTION 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. 1991). 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 identifi- 4 Shaimaa S. Sobieh, Mona H. Darwish cation methods, such as morphological and anatomical observations and microscopic analyses. DNA barcod- ing 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 use- ful to both palaeontologists and evolutionary geneti- cists. This opens the possibility for coordinated studies of macro- and microevolutionary patterns that directly approach the relationship between morphological chang- es 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 sam- ples 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 exist- ence of inhibitors of Taq DNA polymerase in ancient samples, while the third is the small quantity and low quality of DNA that is regained from dead wood (Kaes- tle and Horsburgh 2002) and this is due to degradation of DNA into small fragments in dead tissue (Deguil- loux 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 (Gol- oubinoff et al. 1993). Wagner et al. (2018) character- ized 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 sam- ples, in terms of a pre-existing classification (Tripathi et al. 2013) or to assess whether species should be com- bined 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. 2011). 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 oxy- gen 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 Depart- ment, 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 barcod- ing 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. MATERIAL AND METHODS Population sampling Fossil samples Seven of the good quality Egyptian ancient Mio- cene petrified dicot wood specimens (23.03  to  5.33  Ma. years ago) were used to extract the aDNA. Only two specimens (Bombacoxylon owenii (Bombacaceae) and Dalbergioxylon dicorynioides (Fabaceae) (Fig. 1a, b) were successfully identified to the genus level by the analysis of the ITS of the nuclear ribosomal DNA and the other five samples gave negative results. These Miocene petri- fied dicot woods were found in the Wadi Natrun area in Egypt and were previously identified by palaeobotanists (El-Saadawi et al. 2014; Kamal EL-Din et al. 2015) on the basis of the wood anatomy. The wood specimens were housed in the palaeobotanical collection of the Botany Department, Faculty of Science, Ain Shams University, Cairo-Egypt. 5The first molecular identification of Egyptian Miocene petrified dicot woods (Egyptians’ dream becomes a reality) Nearest Living Relative (NLR) samples Liv ing wood tissue from Bombax ceiba and Dalbergia sissoo was used in the present study as the NLR samples of Bombacoxylon owenii and Dalbergioxylon dicorynioides, respectively. DNA Extraction, Amplification, and Sequencing DNA Extraction Total genomic DNA was extracted from the liv- ing woods and fossil wood using the cetyltrimethylam- monium bromide method (CTAB) described by Doyle and Doyle (1987). As the extraction of aDNA in fossils is more difficult than the extraction of DNA from living wood several modifications were made. Layers of fos- sil surfaces were scraped with a sterile scalpel and were discarded under sterile conditions in order to remove any contamination, and mechanical disruption was used during the DNA extraction procedure. The original fos- sil samples were loose fragments scattered on the sand surface ranging between 10-50 cm in length and 5-20 cm in diameter (El-Saadawi et al., 2014). They were very hard and difficult to break so they were cut by marble cutting machine into pieces and then those pieces were grinded mechanically into fine powder. The starting weight of the fossil sample was five times (5 g) higher than the living wood samples. Three volumes more of extraction buffer than the protocol suggested were add- ed. Polyvinyl pyrrolidone was added to the lysis buffer. The quality of the DNA was estimated by checking the absorbance ratio at 260/280 nm using a Spectronic 21D spectrometer. The DNA samples from both the living and fossil samples were stored at -20°C for amplification and sequencing. DNA Barcode The internal transcribed spacers ITS of the nuclear ribosomal DNA was amplified using ITS4 and ITS5 primers with sequences of ITS4: TCC TCC GCT TAT TGA TAT GC and ITS5: GGA AGT AAA AGT CGT AAC AAG G (White et al. 1990). This region consists of a portion of 18S rDNA, ITS1, 5.8S rDNA, ITS2, and a portion of 28S rDNA (van Nues et al. 1994). The PCR mixture was a 25 μL solution containing 0.5 μL of dNT- Ps (10 mM), 0.5 μL of MgCl2 (25 mM), 5 μL of 5× buffer, 1.25 μLof primer (10 pmol), 0.5 μL of template DNA (50 ng μL–1), 0.1 μL of Taq polymerase (5 U μL–1) and 17.15 μL of sterile ddH2O. The amplification was carried out in a Techni TC-312 PCR, Stafford, UK system. The PCR cycles were programmed for the denaturation process for 4 min at 95°C (one cycle), followed by 30 cycles as follows: 94°C for 1 min; 53°C for 40 s;72°C for 1 min and finally one cycles extension of 72°C for 10 min and 4°C(infinite). The PCR products were run on 1.5% aga- rose gels, which were stained with ethidium bromide, at 120 V for 1 h. Successful PCR products were sent to LGC Genomics Sequencing (Germany) to be sequenced on a 3730xl DNA Analyzer (Applied BiosystemsTM/ Thermo Fisher Scientific). Data analysis The sequence identity was determined using the BlASTn algorithm available through the National Cent- er for Biotechnology Information (NCBI) https://www. ncbi.nlm.nih.gov. The consensus sequences that showed a significant match with the earlier identified data in the NCBI were submitted to the Barcode of Life Data system (BOLD) v4 http://www.barcodinglife.org to identify each sequence sample to the genus and species level. The new fossil sequences were submitted to the NCBI to be listed and recorded in the GenBank data- base. The G+C content of the four samples were calcu- Fig. 1. Sections of Bombacoxylon owenii (a) and Dalbergioxylon dicorynioides (b). (a) (b) 6 Shaimaa S. Sobieh, Mona H. Darwish lated online using the CG content calculator website https://www.biologicscorp.com/tools/GCContent#.WrS- k5OhubIU. The multiple DNA sequences alignments (MSA) were performed using t he Molecu lar Evolution- ary Genetics Analysis version 6 (MEGA 6) (Tamura et al. 2013), while double sequence alignment using the CLUSTAL W algorithm was performed according Thompson et al. (1994). The genetic distances were computed using MEGA 6.06 according to the Kimura-2-Parameter (K2P) model (Kimura 1980). Phylogenetic reconstruction The aligned DNA sequences by the CLUSTAL W algorithm of MEGA 6 were trimmed online using the trimming website: http://users-birc.au.dk/biopv/ php/fabox/alignment_trimmer.php. The final aligned sequences were used to construct the phylogenetic trees. Sixteen species with their accession numbers (Table 1) were used to construct the phylogenetic tree for cf. Ceiba sp., and 36 species with their accession numbers (Table 2) were used to construct the phylogenetic tree for cf. Dalbergia sp. Moreover, the sequences of Persea pseudo- carolinensis (accession number. AY337335) and Persea palustris (accession number. AY3377330) from GenBank, were chosen as outgroup to root the trees. The maximum likelihood (ML) analysis was applied to construct the phylogenetic trees. The ML analy- sis was constructed in MEGA 6 using the K2P model, with 1,000 bootstrap replicates. The codon positions were combined as 1st+2nd+3rd+noncoding. All posi- tions containing gaps and missing data were eliminated. The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to assume the phylogenetic tree. Table 1. The eighteen species used for constructing the phylogenetic tree for cf. Ceiba sp. with their accession numbers. Accession number Corresponding species MG603734 cf. Ceiba sp. KM453172 Ceiba ventricosa KM453167 Ceiba erianthos KM453170 Ceiba pubiflora HQ658387 Ceiba crispiflora KM453171 Ceiba rubriflora HQ658388 Ceiba speciosa KM488629 Ceiba insignis KM453168 Ceiba jasminodora DQ284851 Ceiba pentandra HQ658389 Ceiba schottii HQ658384 Ceiba aesculifolia HQ658385 Ceiba acuminata HQ658376 Bombax buonopozens KM453163 Bombax ceiba DQ826447 Bombax malabaricum AY337335 Persea pseudocarolinensis AY3377330 Persea palustris Table 2. The thirty-eight species used for constructing the phyloge- netic tree for cf. Dalbergia sp. with their accession numbers. Accession number Corresponding species MG450751 cf. Dalbergia sp. KM521409 Dalbergia sissoo KP092712 Dalbergia balansae KM521377 Dalbergia odorifera AB828610 Dalbergia assamica  KM521378 Dalbergia hupeana KM521413 Dalbergia stipulacea AB828616 Dalbergia bintuluensis AB828639 Dalbergia hostilis KM521372 Dalbergia dyeriana AB828619 Dalbergia bracteolata AF068140 Dalbergia congestiflora AB828632 Dalbergia frutescens AB828633 Dalbergia glomerata AB828649 Dalbergia melanocardium KM276143 Dalbergia melanoxylon KM276125 Dalbergia latifolia AB828614 Dalbergia benthamii AB828622 Dalbergia canescens AB828608 Dalbergia arbutifolia AB828626 Dalbergia cultrate AB828605 Dalbergia acariiantha AB828618 Dalbergia bojeri AB828613 Dalbergia baronii AB828640 Dalbergia humbertii AB828635 Dalbergia greveana AB828604 Dalbergia abrahamii KM521415 Dalbergia trichocarpa AB828648 Dalbergia martini FR854138 Dalbergia tonkinensis AB828653 Dalbergia parviflora HG313773 Dalbergia entadoides KM521404 Dalbergia rimosa HG004883 Dalbergia cf. kingiana HG313775 Dalbergia dialoides KM521414 Dalbergia subcymosa AY337335 Persea pseudocarolinensis AY3377330 Persea palustris 7The first molecular identification of Egyptian Miocene petrified dicot woods (Egyptians’ dream becomes a reality) RESULTS AND DISCUSSION DNA isolation As far as is known, this is the first time that DNA from ancient Egyptian wood samples was extracted. The absorbance ratios (A260/280 nm) of the DNA extracts ranged between 1.81- 1.94 (Table 3), indicat- ing good quality of the DNA from both fossil and liv- ing specimens. The concentrations of the DNA extracts were 175,285, 375 and 470 ng/ μL for Dalbergioxylon dicorynioides, Bombacoxylon owenii, Bombax ceiba and Dalbergia sissoo, respectively, as given in Table 3. At the present time, publications of aDNA from plant fossils are still relatively infrequent; however, there are many aDNA publications from animals and humans which make up most samples in this field (Gugerli et al. 2005). Helentjaris (1988) indicated that plant material from archaeological sites may also be amenable to DNA analysis. Many researchers have explored the possibility of isolating DNA from ancient wood samples. DNA has been extracted from samples of modern papyri (writ- ing sheets made with strips from the stem of Cyperus papyrus) varying in age from 0-100 years BP and from ancient specimens from Egypt, with an age-span from 1,300-3,200 years BP. The results showed that the DNA half-life in papyri is approximately 19-24 years. This means that the last DNA fragments will vanish within no more than 532-672 years from the sheets being man- ufactured (Marota et al. 2002). In the case of ancient wood, the risk of contamination during handling and analysis is lower than with human or microbial DNA (Gilbert et al. 2005). Earlier works on fresh wood by Asif and Cannon (2005), Deguilloux et al. (2006) and studies of aDNA from ancient wood from Quercus and Crypto- meria by Deguilloux et al. (2002) suggested the possibil- ity of DNA survival in ancient wood remains, which was confirmed by the current work. Liepelt et al. (2006) reported that, it was possible to isolate DNA from wood as old as 1000 years. Depending on the mode of conservation and the climate at the exca- vation site, as well older samples could be isolated and analysed successfully (Deguilloux et al. 2006). DNA Barcoding by ITS The DNA barcoding affords an important step for the molecular identification of aDNA from petrified woods. The amplification of genomic DNA uses the uni- versal primers for the ITS region. Two of seven aDNA extracts from the dicot wood fossil samples (Bombacoxylon owenii and Dalbergioxylon dicorynioides) were successfully used to amplify the ITS region. The PCR and sequencing success rates for the fossil and living samples were 100% (Table 4). The genus and species identity results of the query sequences were then determined using the BLAST and BOLD databases to estimate the reliability of the genus identification. The results of both databases showed that ITS was 100% cor- rectly identified at the genus level, while the success rates for species identification were 50 and 25% for BLAST and BOLD respectively (Table 5). Many studies have compared the discriminatory power revealed by the ITS region in its entirety with Table 3. Optical densities and concentrations of the DNA isolated from fossil and living specimens. Plant name Optical density Ratio 260/280 nm DNA concentration (ng/µL)260 nm 280 nm Bombacoxylon owenii 0.057 0.032 1.84 285 Bombax ceiba 0.075 0.041 1.82 375 Dalbergioxylon dicorynioides 0.035 0.018 1.94 175 Dalbergia sissoo 0.094 0.052 1.81 470 Table 4. Success rates of the amplification and sequencing. Barcode locus Number of tested samples (fossil and living samples) No of samples amplified and percentage of PCR success Number and percentage of PCR failure Number and percentage of sequencing success ITS 4 4 (100%) 0 (0%) 4 (100%) 8 Shaimaa S. Sobieh, Mona H. Darwish ITS2, proposing the use of ITS2 as an alternative bar- code to the entire ITS region (Han et al 2013). ITS2 was previously used as a standard DNA barcode to identify medicinal plants by Chen et al. (2010) and a barcode to identify animals (Li et al 2010). The length of the ITS2 region is sufficiently short to allow for the easy amplifi- cation of even degraded DNA, and the ITS2 region has enough variability to distinguish even closely related species and has conserved regions for designing univer- sal primers (Yao et al. 2010). Therefore, it could be used as a DNA barcode for plant fossils in further investiga- tions. In addition, all 4 raw nucleotide sequences were verified with the other available sequences in Gen- Bank using the BLASTn algorithm. The sequences of the two living samples of Bombax ceiba and Dalbergia sissoo showed an identity ratio of 99% with Bombax ceiba (accession no. KM453163) and Dalbergia sissoo (accession no. AB828659), respectively (Table 6). The identification of the fossil samples: Based on the author’s knowledge, thus far, there has been no published work on aDNA from petrified wood. Therefore, this is considered the first molecular identi- fication of Egyptian plant fossil remains and of petri- fied wood (Bombacoxylon owenii and Dalbergioxylon dicorynioides) worldwide. Meanwhile, the authors hope that many other fields (anatomy and morpholog y) besides the molecular field will contribute to determin- ing the relationship between living plants and their fossil remains. Bombacoxylon owenii (cf. Ceiba sp. accession no.: MG603734) The ITS sequence from the fossil specimen was amplif ied and produced a 704 bp fragment. The sequence was uploaded to the NCBI database and was documented, for the first time with accession number MG603734. Bombacoxylon owenii was listed in the NCBI data- base as cf. Ceiba sp. because the GenBank policy is not to add fossil taxa to the taxonomy database, since it is a database of living or recently extinct organisms. Bombacoxylon is a fossil genus for woods with fea- tures characteristic of the Bombacoideae, not a whole plant. Moreover, the molecular identification revealed a close resemblance of the submitted sequence to Ceiba pentandra (the commercial kapok tree) rather than Bombax as was expected by Kamal El-Din et al. (2015). This identification is not surprising since the two living genera (Bombax and Ceiba) are grouped in the same subfamily Bombacoideae and have very few differences between them. Moreover, the wood anatomy of both genera reveals the high resemblance between them, and they can be only distinguished by a combination of macroscopic characteristics, which are the shape of the vessel-ray pit, the ray width, the sheath cells and mineral inclusion (Nordahlia et al., 2016). The NLR of some fossil wood taxa might be wrong, Bombacoxylon shares characters with Sterculi- aceae and Bombacaceae rather than only with Bombax, Grewioxylon with other members of the Malvaceae with tile cells, (e.g., Craigia) instead of only Grewia (Skala 2007). In addition, Wickens (2008) stated that it must Table 5. Identification efficiency of the barcode loci using BLAST and BOLD. Barcode Locus No. of samples identified Family level using BLAST Family level using BOLD Genus level using BLAST Genus level using BOLD Species level using BLAST Species level using BOLD ITS  4 100% 100% 100% 100% 50% 25% Table 6. Identification matches of the ITS sequences using the BLAST and BOLD Databases. Sample identification Plant order Plant family Plant subfamily BLAST search match BLAST similarity (%) BOLD search match BOLD similarity (%) cf. Ceiba sp. (Bombacoxylon owenii) Malvales Malvaceae Bombacoideae cf. Ceiba sp. 100 Ceiba pantandra 90.83 Bombax ceiba Malvales Malvaceae Bombacoideae Bombax ceiba 99 Bombax malabaricum 99.14 cf. Dalbergia sp. (Dalbergioxylon dicorynioides) Fabales Fabaceae Papilionoideae cf. Dalbergia sp. 100 Dalbergia odorifera 87.94 Dalbergia sissoo Fabales Fabaceae Papilionoideae Dalbergia sissoo 99 Dalbergia sissoo 98.57 9The first molecular identification of Egyptian Miocene petrified dicot woods (Egyptians’ dream becomes a reality) not be assumed that the names of fossil wood necessar- ily represent species close to modern genera. The sequence of cf. Ceiba sp. was compared with other available sequences in GenBank using the BLASTn algorithm. The results showed that the sequences belonged to the homologous sequences of the genus Ceiba. The sequence of cf. Ceiba sp. showed identities with several living Ceiba species rather than Bombax. The identity ratios among the Ceiba species indicated that the Ceiba pentandra ITS nucleotide sequence (acces- sion no. DQ284851) was the nearest related ITS sequence for Bombacoxylon owenii (cf. Ceiba sp.). The sequence of cf. Ceiba sp. was aligned with both Bombax ceiba (accession no. KM453163) and Ceiba pentandra (accession no. DQ284851) using CLUSTAL W (Thompson et al. 1994). The identity between the cf. Ceiba sp. ITS sequence and that of Ceiba pentandra was 625 (80.23%) (Fig. 2a), while the identity between the cf. Ceiba sp. ITS sequence and that of Bombax ceiba was 548 (76.54%) (Fig. 2b). The final aligned sequences obtained by sequence trimming revealed that G+C content was obviously higher than of A+T content (Table 7). Genetic distances were calculated by the Kimura-2-Parameter (K2P) model (Kimura 1980). Moreover, both Bombacoxylon owenii (cf. Ceiba sp.) and Ceiba pentandra shared similarities in the wood anatomy characteristics, with the presence of diffuse to semiring porous wood in both of them. Bombacoxylon owenii (cf. Ceiba sp.) and Ceiba pentandra contain soli- tary vessels and have radial multiples of 2 to 4 and medi- um to large vessels that are often filled with tyloses. The growth rings in both are distinct or absent and the ves- sel frequency is 5 to 20 per mm2. The perforation plates are simple, and the intervessel pits are alternate. The ves- sel-ray parenchyma pits are like the intervessel pits and the fibres are nonseptate with thick-walls and diffuse to diffuse-in-aggregate axial parenchyma (Table 8) (inside wood 2013; Kamal EL-Din et al. 2015; Nordahlia et al. 2016). Dalbergioxylon dicorynioides (cf. Dalbergia sp.accession no.: MG450751) The ITS sequence (610 bp) was amplified and record- ed in the NCBI database with GenBank accession no. MG450751. Dalbergioxylon dicorynioides was recorded as cf. Dalbergia sp. in the NCBI, since it is  a database of living or recently extinct organisms. Dalbergioxy- lon dicorynioides is a fossil genus for woods not a whole plant. Fig. 2. (a) Sequence alignment between cf. Ceiba sp. and Ceiba pentandra (accession no. DQ284851) using CLUSTAL W, Identity (*): 625 is 80.23 %. (b) Sequence alignment between cf. Ceiba sp. and Bombax ceiba (accession no. KM453163 ) using CLUSTAL W, Identity (*): 548 is 76.54 %. (a) (b) 10 Shaimaa S. Sobieh, Mona H. Darwish The total sequence length of ITS in the Dalbergia genus ranged from 600 to 800 bp as reported by several records in the NCBI database for ITS in the Dalbergia genus. The sequence was tested with other available sequences in GenBank using the BLASTn algorithm. The results showed that the sequences belonged to the homologous sequences of the genus Dalbergia. The sequence of cf. Dalbergia sp. showed identities with sev- eral living Dalbergia species, but when we compared the identity ratios among them we found that the Dal- bergia sissoo ITS nucleotide sequences (accession no. AB828659.1) were the nearest ITS sequence for Dalbergi- oxylon dicorynioides (cf. Dalbergia sp.), with an identity ratio of 91%. The final aligned sequences obtained by sequence trimming revealed that the G+C content was obviously higher than the A+T content (Table 7). Genetic distances for Dalbergia sequences alignment were calculated by the Kimura-2-Parameter (K2P) model (Kimura 1980). The comparison of the wood anatomy characteris- tics of Dalbergioxylon dicorynioides (cf. Dalbergia sp.) with those of living Dalbergia species revealed that Dal- bergia sissoo was most closely related to Dalbergioxylon dicorynioides (Table 9) because both contained diffuse- porous wood, solitary vessels and radial multiples of 2 to 3, indistinct or absent growth rings, exclusively sim- ple perforation plates, alternate and vestured intervessel pits, vessel-ray pits similar to intervessel pits in size and shape throughout the ray cell, combinations of aliform, confluent and irregular banded (1 to 4 cells wide) axial parenchyma, 1-3 seriate rays up to 20 cells high, and thick-walled non-septate fibers (inside wood 2013; El- Saadawi et al. 2014). Phylogenetic analysis The phylogenetic ana lyses were conducted in MEGA6 (Thompson et al. 1994) and the phylogenetic trees were inferred with the ML based on the Kimura model (Kimura 1980). Nowadays, several programs can be used to construct maximum likelihood phylogenetic tree. The fastest ML-based phylogenetic programs that differ in implementations of rearrangement algorithms are PhyML (Guindon et al. 2010) and RAxML/ExaML (Stamatakis 2014). The topologies of the phylogenetic trees were evalu- ated using the bootstrap resampling method of Felsen- stein (1985) with 1000 replicates. The analysis involved Table 7. Sequence length and GC and AT content. Sample name Full length G+C G+C% A+T A+T% cf. Ceiba sp. 704 221+219 62% 134+130 38% Bombax ceiba 692 231+234 66% 113+114 34% cf. Dalbergia sp. 610 185+194 61% 135+96 39% Dalbergia sissoo 610 189+211 64% 127+83 36% Table 8. Comparison of anatomical features between Bombacoxylon owenii & Ceiba pentandra. Species Feature Bombacoxylon owenii Ceiba pentandra (L.) Growth ring Distinct Distinct, indistinct or absent Porosity Diffuse to semiring-porous Diffuse-porous Perforation plates Simple Simple Intervessel pits Alternate Alternate Radial diameter 240 μm (220 to260) 350 to 800 µm Vessels groupings Solitary and in radial multiples of 2 to 4  Restricted to marginal rows Tyloses Common  Common Vessel/mm2 5 to 15(8) 5 to 20 Vessel element length μm 335 μm 350 to 800 µm Axial Parenchyma Diffuse, diffuse-in-aggregates, scanty, narrow vasicentric paratracheal and in narrow bands or lines Diffuse, diffuse-in-aggregates, scanty, narrow vasicentric paratracheal and in narrow bands or lines Rays 1 to 3 cells, seriate Larger rays commonly 4 to 10 seriate Fibers Nonseptate with very thick walls Nonseptate with thin- to thick-walled 11The first molecular identification of Egyptian Miocene petrified dicot woods (Egyptians’ dream becomes a reality) 18 nucleotide sequences (cf. Ceiba sp., 12 species of Ceiba and 3 species of Bombax which were downloaded from the NCBI database), and Persea pseudocarolinensis and Persea palustris  were used as outgroups. There was a total of 1374 positions in the final dataset, and the ambiguous positions were completely eliminated for each sequence pair. The ML tree was divided into two clades, namely A and B. Clade A included Bombax members, while clade B included the Ceiba species in addition to cf. Ceiba sp. (Bombacoxylon owenii). Both cf. Ceiba sp. and Ceiba pentandra were on the same branch. Therefore, the phylogenetic tree showed that Bombacoxylon owenii (cf. Ceiba sp.) was very similar to the Ceiba genus, which previously was thought to resemble the Bombax genus (Kamal EL-Din et al. 2015) (Fig. 3). In the ML tree, all the Dalbergia species were divid- ed into two clades, namely clade A and clade B (Fig. 4). Clade A includes cf. Dalbergia sp. and Dalbergia sissoo. The second group (clade B) was subdivided into many subclades that contained the other species of Dalbergia. Therefore, the present work matches the palaeobotanist Table 9. Comparison of anatomical features between Dalbergioxylon dicorynioides & Dalbergia sissoo. Species Feature Dalbergioxlon dicorynioides Dalbergia sissoo Growth ring Absent. Distinct, indistinct or absent Porosity Diffuse- porous Diffuse- porous Perforation plates Simple Simple Intervessel pits Alternate Alternate Tangential diameter μm 170 μm (range 100 to 210 μm) 100 to 200 Vessels groupings Solitary and in radial multiples 2 to 3 Solitary or grouped in radial multiples of 2 to3 cells. Vessel groupings /mm2 8/mm2 (range 5 to13/ mm2) 5 to 20  Vessel element length μm 330 μm (range 280 to 410 μm) <= 350 Axial Parenchyma Aliform, confluent and irregular banded (1 to 4 celled wide) Aliform, confluent and irregular banded, 4 (3to 4) cells per parenchyma strand Rays 1to 3 seriate 1 to 3 cells Fibers Thick-walled, nonseptate Very thick-walled, nonseptate  Fig. 3. Maxim- Likelihood (ML) cladogram showing the relation- ships of the ITS gene from cf. Ceiba sp. in relation to its relatives. All analyses were performed with 1000 bootstrap replicates (arrow: fossil specimens, acc. no.: accession number). Fig. 4. Maxim- Likelihood (ML) cladogram showing the relation- ships of the ITS gene from cf. Dalbergia sp. in relation to its rela- tives. All analyses were performed with 1000 bootstrap replicates (arrow: fossil specimens, acc. no.: accession number). 12 Shaimaa S. Sobieh, Mona H. Darwish assumption that there is a close relationship between Dalbergioxylon dicorynioides (cf. Dalbergia sp.) and Dal- bergia sissoo. CONCLUSION The DNA barcoding dataset in the present study provides an important first step towards establish- ing an effective molecular tool for the identification of aDNA from petrified woods. We hope that these results will encourage reliable aDNA studies of other petrified woods. The further studies of ancient wood DNA from the abundant store of fossil plant remains will rely on this study and by the intensive works of researchers from different fields, and these findings could provide a powerful tool to increase world knowledge about the history of forests, plant evolution and historical bioge- ography. AUTHOR CONTRIBUTIONS Both authors suggested the point of the work and Dr. Shaimaa S. Sobieh planned the experimental design to achieve this point. Both authors supplied the financial support for the work. Prof. Mona Darwish shared other palaeobotanists in the identification of dicot woods (see El-Saadawi et al. 2014). The experimental part was done by Dr/Shaimaa S. Sobieh. The writing of the manuscript was done by both authors. ACKNOWLEDGEMENTS The authors would like to thank Prof. Wagih El- Saadawi and Prof. Marwa Kamal El-Din (Profs. of Pal- aeobotany, Botany Department, Faculty of Science, Ain Shams University) for supplying them the fossil speci- mens. In addition, they thank Fatma Abdel Naby Mursi and Aya Abdel Gawad (MSc. students, Botany Depart- ment, Faculty of Women for Art, Science and Education, Ain Shams University) for helping them in the extrac- tion of aDNA. Finally, the authors would like to thank Dr Enas Hamdy Ghallab (Lecturer of Medical Entomol- ogy, Entomology Department, Faculty of Science, Ain Shams University) and Mohamed Emad El-din Elsaid (Biotechnology Bachelors, Misr University for Science and technology) for helping the authors in understand many points in the bioinformatics programs. REFERENCES Asif MJ, Cannon CH. 2005. DNA extraction from pro- cessed wood: a case study for the identification of an endangered timber species (Gonstylus bancanus). Plant Mol Biol Rep. 23:1–8. Chen S, Yao H, Han J, Liu C, Song J, Shi L, et al .2010. Validation of the ITS2 region as a novel DNA bar- code for identifying medicinal plant species. PLoS ONE 5: e8613. doi.org/10.1371/journal.pone.0008613 Deguilloux MF, Bertel L, Celant A, Pemonge MH, Sadori L, Magri D, Petit RJ. 2006. Genetic analysis of archae- ological wood remains: first results and prospects, J Archaeol Sci. 33: 1216–1227. Deguilloux MF, Petit MH, Pemonge RJ. 2002. Novel perspectives in wood certification and forensics: dry wood as a source of DNA. Proc R Soc London. 269: 1039-1046. Doyle JJ, Doyle JL. 1987. A rapid DNA isolation proce- dure for small quantities of fresh leaf tissue. Phytoch Bull. 19: 11-15. El-Saadawi W, Kamal El-Din MM, Darwish MH, Osman R. 2014. African Miocene dicot woods with two new records for this epoch from Egypt. Taeckholmia. 34:1-2. Felsenstein J. 1985. Confidence limits on phylogenies: An approach using the bootstrap. Evol. 39: 783-791. Gilbert MTP, Bandelt HJ, Hofreiter M, Barnes I. 2005. Assessing ancient DNA studies. Tren Ecol Evol. 20: 541–544. Golberg EM, Brown TA, Bada JL, Westbroek P, Bishop MJ, Dover GA. 1991. Amplification and analysis of Miocene plant fossil DNA. Phil Trans R Soc Lond B. 333: 419-427. Golenberg EM. 1994. Fossil samples DNA from plant compression fossils. In: Herrmann B, Hummel S, (eds) Ancient DNA recovery and analysis of genetic material from paleontological, archaeological, muse- um, medical, and forensic specimens. New York: Springer-Verlag Inc pp 237-256. Goloubinoff P, Pääbo S, Wilson A. 1993. Evolution of Maize Inferred from Sequence Diversity of an adh2 Gene Segment from Archaeological Specimens. Proc Natl Acad Sci USA. 90:1997-2001. Gugerli F, Parducci L, Petit RJ. 2005. Ancient plant DNA: review and prospects, New Phytologist. 166: 409–418. Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O. 2010. New algorithms and methods to estimate maximumlikelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol. 59(3): 307–321. Gyulai G, Humphreys M, Lagler R, Szabó Z, Tóth Z, Bittsánszky A, Gyulai F, Heszky L. 2006. Seed 13The first molecular identification of Egyptian Miocene petrified dicot woods (Egyptians’ dream becomes a reality) remains of common millet from the 4th (Mongolia) and 15th (Hungary) Centuries: AFLP, SSR and mtD- NA sequence recoveries. Seed Sci Res. 16: 179-191. Han J, Zhu Y, Chen X, Liao B, Yao H, Song J, Chen S, et al. 2013. The short ITS2 sequence serves as an effi- cient taxonomic sequence tag in comparison with the full-length ITS. Biomed Res Int. 2013:741476. doi. org/10.1155/2013/741476 Hamalton T. 2016. DNA from ancient wood. Van Sang- yan. 3: 27-30. Helentjaris T. 1988. Maize Genet Coop. News Lett. 62: 104-105. Hollingsworth PM, Graham SW, Little DP. 2011. Choos- ing and using a plant DNA barcode. PLoS ONE. 6: e19254. doi.org/10.1371/journal.pone.0019254 Kaestle AF, Horsburgh KA. 2002. Ancient DNA in anthropology: methods, applications, and ethics. Am J Phys Anthropol. 35: 92-130. Kamal EL-Din MM, Darwish MH, EL-Saadawi W. 2015. Novelties on Miocene woods from Egypt with a sum- mary on African fossil woods of Fabaceae, Malva- ceae and Dipterocarpaceae. Palaeontographica Abt B. 292:173-199. Kim S, Soltis DE, Soltis PS, Suh Y. 2004. DNA sequences from Miocene fossils: an ndhF sequence of Magno- lia latahensis (Magnoliaceae) and an rbcl sequence of Persea pseudocarolinensis (Lauraceae). Am J Bot . 91: 615–620. Kimura M. 1980. A simple method for estimating evolu- tionary rate of base substitutions through compara- tive studies of nucleotide sequences. J Mol Evol. 16: 111-120. Liepelt S, Sperisen C, Deguilloux MF, Petit RJ, Kissling R, Spencer M, De Beaulieu J, Taberlet P, Gielly l, Zie- genhagen B. 2006. Authenticated DNA from Ancient Wood Remains. Ann Bot. 98: 1107–1111. Li YW, Zhou X, Feng G, Hu HY, Niu LM, Hebert PD, et al. 2010. COI and ITS2 sequences delimit species, reveal cryptic taxa and host specificity of fig-associat- ed Sycophila (Hymenoptera, Eurytomidae). Mol Ecol Resour 10: 31–40. Marota I, Basile C, Ubaldi M, Rollo F. 2002. DNA decay rate in Papyri and human remains from Egyptian archaeo- logical sites. Am j phys anthropol. 117: 310–318. Nasab HM, Mardi M, Talaee H, Nashli HF, Pirseyedi SM, Nobari AH, Mowla SJ. 2010. Molecular analysis of ancient DNA extracted from 3250-3450 year-old plant seeds excavated from Tepe Sagz Abad in Iran. J Agr Sci Tech. 12: 459-470. Nordahlia AS, Noraini T, Chung RCK, Lim SC, Nadiah I, Azahana NA, Solihani NS. 2016. Comparative wood anatomy of three Bombax species and Ceiba pentandra (Malvaceae: Bombacoideae) in Malaysia. Mal Nat J. 68: 203-216. Poinar HN, Cano RJ, Poinar GO. 1993. DNA from an extinct plant. Nature 363: 677. Rogers SO, Bendich AJ. 1985. Extraction of DNA from milligram amounts of fresh, herbarium and mummi- fied plant tissues. Pl molec Biol. 5: 69-76. Sakala, J. 2007. The potential of fossil angiosperm wood to reconstruct the palaeoclimate in the Tertiary of Central Europe (Czech Republic, Germany). Acta Palaeobotanica.. 47: 127–133 (2007). Stamatakis A. 2014. RAxML version 8: a tool for phyloge- netic analysis and post-analysis of large phylogenies. Bioinformatics. 30(9):1312–1313. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol. 30: 2725-2729. Thompson JD, Higgins DG, Gibson TG. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucl Aci Res. 22: 4673-4680. Tripathi AM, Tyagi A, Kumar A, Singh A, Singh S, Chaudhary LB, Roy S. 2013. The internal transcribed spacer (ITS) region and trnhHpsbA are suitable can- didate loci for DNA barcoding of tropical tree species of India. PloS ONE. 8: e57934. doi.org/10.1371/jour- nal.pone.0057934. van Nues R W, Rientjes J M J, van der Sande C A F M., Zerp S F, Sluiter C, Venema J, Planta R J, Raue´ HA. 1994. Separate structural elements within internal transcribed spacer 1 of Saccharomyces cerevisiae pre- cursor ribosomal RNA direct the formation of 17S and 26S rRNA. Nucl Aci Res. 22: 912–919. Wagner S, Lagane F, Seguin-Orlando A, et al. 2018 High- Throughput DNA sequencing of ancient wood. Mol Ecol. 27: 1138-1154. White TJ, Bruns TD, Lee SB, Taylor JW. 1990. Amplifi- cation and direct sequencing of fungal ribosomal RNAgenes for phylogenetics. In: Innis MA, Gelfard H, Sninsky JS, WhiteTJ (eds) PCR-protocols and applications. A laboratory manual. New York: Aca- demic Press, pp 315–322. Wickens GE. 2008. The Baobabs: Pachycauls of Africa, Madagascar and Australia Springer Science & Busi- ness Media  Wood data base available at inside wood home page. 2013. Online search of fossil and modern. Yao H, Song J, Liu C, Luo K, Han J, Li Y, et al. 2010. Use of IRS2 region as the universal DNA barcode for plants and animals. PLoS ONE 5: e13102. doi. org/10.1371/journal.pone.0013102 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Volume 73, Issue 2 - 2020 Firenze University Press The first molecular identification of Egyptian Miocene petrified dicot woods (Egyptians’ dream becomes a reality) Shaimaa S. Sobieh*, Mona H. Darwish Gene flow patterns reinforce the ecological plasticity of Tropidurus hispidus (Squamata: Tropiduridae) Fernanda Ito, Danielle J. Gama-Maia, Diego M. A. Brito, Rodrigo A. Torres* The technique of Plant DNA Barcoding: potential application in floriculture Antonio Giovino1,*, Federico Martinelli2,*, Anna Perrone3 Cytogenetic of Brachyura (Decapoda): testing technical aspects for obtaining metaphase chromosomes in six mangrove crab species Alessio Iannucci1, Stefano Cannicci1,2,*, Zhongyang Lin3, Karen WY Yuen3, Claudio Ciofi1, Roscoe Stanyon1, Sara Fratini1 Comparison of the Evolution of Orchids with that of Bats Antonio Lima-de-Faria Identification of the differentially expressed genes of wheat genotypes in response to powdery mildew infection Mehdi Zahravi1,*, Panthea Vosough-Mohebbi2, Mehdi Changizi3, Shahab Khaghani1, Zahra-Sadat Shobbar4 Populations genetic study of the medicinal species Plantago afra L. (Plantaginaceae) Saeed Mohsenzadeh*, Masoud Sheidai, Fahimeh Koohdar A comparative karyo-morphometric analysis of Indian landraces of Sesamum indicum using EMA-giemsa and fluorochrome banding Timir Baran Jha1,*, Partha Sarathi Saha2, Sumita Jha2 Chromosome count, male meiotic behaviour and pollen fertility analysis in Agropyron thomsonii Hook.f. and Elymus nutans Griseb. (Triticeae: Poaceae) from Western Himalaya, India Harminder Singh2, Jaswant Singh1,*, Puneet Kumar2, Vijay Kumar Singhal1, Bhupendra Singh Kholia2, Lalit Mohan Tewari3 Population genetic and phylogeographic analyses of Ziziphora clinopodioides Lam., (Lamiaceae), “kakuti-e kuhi”: An attempt to delimit its subspecies Raheleh Tabaripour1,*, Masoud Sheidai1, Seyed Mehdi Talebi2, Zahra Noormohammadi3 Induced cytomictic crosstalk behaviour among micro-meiocytes of Cyamopsis tetragonoloba (L.) Taub. (cluster bean): Reasons and repercussions Girjesh Kumar, Shefali Singh* Karyotype diversity of stingless bees of the genus Frieseomelitta (Hymenoptera, Apidae, Meliponini) Renan Monteiro do Nascimento1, Antonio Freire Carvalho1, Weyder Cristiano Santana2, Adriane Barth3, Marco Antonio Costa1,* Karyotype studies on the genus Origanum L. (Lamiaceae) species and some hybrids defining homoploidy Esra Martin1, Tuncay Dirmenci2,*, Turan Arabaci3, Türker Yazici2, Taner Özcan2 Determination of phenolic compounds and evaluation of cytotoxicity in Plectranthus barbatus using the Allium cepa test Kássia Cauana Trapp1, Carmine Aparecida Lenz Hister1, H. Dail Laughinghouse IV2,*, Aline Augusti Boligon1, Solange Bosio Tedesco1