Advances in Anthropology 2013. Vol.3, No.4, 240-248 Published Online November 2013 in SciRes (http://www.scirp.org/journal/aa) http://dx.doi.org/10.4236/aa.2013.34034 Open Access 240 Identification of Plant Remains in Underwater Archaeological Areas by Morphological Analysis and DNA Barcoding Angelo Gismondi 1 , Donatella Leonardi 1 , Flavio Enei 2 , Antonella Canini 1* 1 Department of Biology, University of Rome “Tor Vergata”, Via della Ricerca Scientifica snc, Rome, Italy 2 Civic Museum and Castle of Santa Severa, Via del Castello snc, Santa Severa, Rome, Italy Email: * canini@uniroma2.it Received May 28 th , 2013; revised June 27 th , 2013; accepted July 29 th , 2013 Copyright © 2013 Angelo Gismondi et al. This is an open access article distributed under the Creative Com- mons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, pro- vided the original work is properly cited. DNA barcode technique has only recently been applied to archaeobotanical studies. In fact, in association with morphological, scanning electron and optical microscopic analyses, these specific methods allow re- searcher to scientifically classify antique flora samples. Therefore, this project wants to improve, to en- courage and spread further use of this protocol and to highlight the potentialities of the molecular biology and microscopy related to botanical fossils. In conclusion, ancient Olea europaea L. and Crataegus monogyna Jacq. seeds, a Pinus sp. pollen cone, a Quercus petraea (Mattuschka) Liebl. acorn, animal fi- bers and gymnosperm woody fragments, found in a 1st Century BC sunken Dressel 1B amphora, have clearly been identified, in order to enhance knowledge about Central Italy past human activity and envi- ronment. This research has also demonstrated the applicability of this scientific approach on specimens derived from underwater archaeological site. Keywords: Barcoding; Optical Microscopy; SEM Analysis; Archaeobotany; Plant Remains Introduction Sea level variations, due to the effects of post-glacial ages and earth’s structure modifications, caused the submersion, or the sinking, of prehistoric and archaeological sites in water (Bailey & Flemming, 2008). The detection of these areas and their great historical interest induced the development of the “underwater archaeology”. On the other hand, this discipline was also encouraged by the discovery of thousands of sunken wrecks and their content. In literature, only a few works are pre- sented about this science, although it produces useful elements to clarify, suppose and predict different aspects of human an- cestors’ activities and ecosystem (Willcox, 1977; Edge & Gib- bins, 1988; Gorham & Bryant, 2001; Hansson & Foley, 2008; Claesson, 2011). Since the beginning of the last century, flora ancient remains have captured the attention of scientists be- cause of the large amount of new information they could pro- vide about the past (Banning, 2002). A lot of vegetal remains were found in archaeological sites; in fact, flowers, leaves, fruits and woods were regularly used as foods, drugs or funeral offers by ancient populations (Grasso & Fiorentino, 2009). The detection of botanical elements in caves, archaeological areas and hypogean structures and their taxonomic identification allowed scientists to increase knowledge on plant evolution, ethnobotany and reconstruction of past environments (Marota et al., 2002; Liepelt et al., 2006). Morphological observation is the principal approach applied by archaeobotanists to classify plant remains; however, this method sometimes showed wrong and contradictory results (Gismondi et al., 2012). On the other hand, the introduction and the application of molecular analyses on ancient remains, especially after the development of next-generation sequencing techniques, allowed researchers to obtain more accurate data, even in presence of scarce amounts of template (Manen et al., 2003). In particular, DNA barcoding, a method that uses standard nucleotide sequence analysis for species classification (Kress & Erickson, 2008), was recently and successfully associated to archaeobotany (Gould et al., 2010; Gismondi et al., 2012). In 1994, at seven meters under- water, a Dressel 1B amphora, dated back to the 1st Century BC, was found on the sandy seabed of the Pyrgi seaport-canal ar- chaeological site (Santa Severa, Rome) (Enei, 2008). Aim of this work was the botanical identification of flora remains, found in Pyrgi archaeological find, applying, but also encour- aging and improving, the combined use of innovative and clas- sical scientific approaches, in order to increase knowledge about past human populations and ecology. Materials and Methods Detection and Cleaning of Plant Remains Vegetal remains were collected from a sealed amphora (type Dressel B1) found underwater in Pyrgi seaport-canal (Santa Severa, Rome) (Enei, 2008). Samples were preserved water- logged until the analyses. Then, they were washed in H 2 Odd, dried at room temperature for 48 hours, decontaminated by UV light for 30 minutes and stored at −20˚C to reduce further an- cient DNA (aDNA) degradation (Poinar, 2002; Pruvost et al., 2007). * Corresponding author.