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 Gismondi1, Donatella Leonardi1, Flavio Enei2, Antonella Canini1*
1Department of Biology, University of Rome “Tor Vergata”, Via della Ricerca Scientifica snc, Rome, Italy
2Civic Museum and Castle of Santa Severa, Via del Castello snc, Santa Severa, Rome, Italy
Received May 28th, 2013; revised June 27th, 2013; accepted July 29th, 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 H2Odd,
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.

A. GISMONDI ET AL.
Morphological Analysis
Samples were documented by a Fujifilm Finepix S1000fd
camera. Morphological observations were performed by a scan-
ning electron microscope (SEM) Leika/LEO stereoscan 440
(25-1000X enlargement) and a light microscope (LM) Nikon
Eclipse E100 (10-40X enlargement). Woody remain sections
were manually carried out by a scalpel.
aDNA Extraction and Contamination Prevention
aDNA was extracted according to Gismondi et al. (2012)
method. Flora remains were grinded up by a domestic grinder
and then further with pestle, mortar and liquid nitrogen. Sam-
ples were resuspended in 0.8 mL of extraction buffer (10 mM
Tris-HCl pH 8; 100 mM EDTA pH 8; 0.5% SDS; 20 µg/mL
Ribonuclease A, RNase Sigma-Aldrich, Italy) and incubated at
37˚C for 1 hour. Samples were kept at 37˚C for other 3 hours,
after addition of proteinase K (100 µg/mL, Sigma-Aldrich). A
volume of cold phenol:chloroform (1:1, pH 8) was added to
each sample; then, tubes were centrifuged at 12.000 rpm/min
for 5 minutes (min) at 4˚C. 0.4 mL of CTAB solution (35 mM
cetyl trimethylammonium bromide; 100 mM NaCl) was added
to supernatants that were also incubated at room temperature
for 1 hour in agitation. Two phenol:chloroform (1:1; pH 8)
extractions were performed further. 100 µL of 2 M NaCl and 1
mL of cold 2-propanol were used to precipitate aDNA collected
in the aqueous phase of each sample. After incubation at −80˚C
over night, samples were centrifuged at 12.000 rpm/min for 30
min at 4˚C. Pellets were resuspended in 100 µL of TE buffer
(10 mM Tris-HCl pH 8; 1mM EDTA pH 8). A sample incuba-
tion, of 20 min at 37˚C, was finally performed with 40 µg
RNase A. All samples were stored at −20˚C. Naturally, all pre-
cautions necessary for preventing sample contaminations dur-
ing the analyses (DNA extraction and PCR reactions) were
taken, as recommended by Willerslev and Cooper (2004), Yang
and Watt (2005) and Gismondi et al. (2012). In particular:
aDNA was extracted in a specific laboratory where modern
DNA was never processed; PCR amplifications were performed
in a physically separated areas; each PCR amplification and
sequencing analysis was repeated, in triplicate, using different
PCR thermocyclers positioned in different laboratories; positive
controls weren’t performed in order to avoid contamination risk,
instead of negative ones that were always carried out; instru-
ments were always cleaned and decontaminated by UV, after
their use.
PCR Amplifications and Target Genes
Barcode gene amplifications were carried out in a final vol-
ume of 50 µL. Each one contained: 1 - 2 µL (of the total extrac-
tion volume) aDNA, 1.5 U RBC Taq DNA Polymerase (RBC
Bioscience), 20 µM of each primers (described in detail in Ta-
ble 1), 0.2 mM of each dNTP (Sigma-Aldrich), 1X RBC Taq
DNA Polymerase buffer (RBC Bioscience), 3 mM MgCl2 and
5% DMSO. Amplification were performed in a IQ5 thermocy-
cler (Biorad, Italy), using the following cycling profile: 4 min at
95˚C; 50 cycles of 30 sec at 95˚C, 30 sec at annealing tempera-
ture (Table 1) and 1 min at 72˚C; 15 min at 72˚C. In this study,
following target genes were chosen: ribulose 1,5-bisphosphate
carboxylase/oxygenase large subunit (RuBisCO large subunit,
rbcL) gene + maturase K (matK) gene + intragenic spacer be-
tween tRNAHisGUG gene and photosystem II thylakoid mem-
brane protein of Mr 32,000 gene (trnH-psbA) (Chase et al.,
2007; Kress & Erickson, 2007; Group CPW, 2009; Seberg &
Petersen, 2009). For each barcode gene, more regions were
amplified, using different primer pairs (F1-R1, F2-R2, F1-R2,
F1-R3 or F3-R3, as reported in Table 1), according to Schlum-
baum et al. (2008) and Gismondi et al. (2012).
Agarose Gel Electrophoresis
PCR products were separated on 1.5% agarose gel containing
10 mg/mL ethidium bromide. The electrophoresis was per-
formed using 1X TAE buffer (40 mM Tris; 1 mM EDTA; 20
mM acetic acid; pH 8.5) and DNA fragments were visualized
under UV light (VersaDoc 4000 MP, BIO-RAD).
Sequencing and D ata Analysis
3 µL of PCR products were subjected to ExoSAP-IT (Affly-
metrix) treatment for 15 min at 37˚C. Then, samples were sub-
jected to PCR (25 cycles of denaturation at 96˚C for 10 sec,
annealing 50˚C for 5 sec and final extension at 64˚C for 4 min),
after addition of 1.5 µL of BigDye (Applied Biosystems) and
1.5 µL of forward (or reverse) primer. Amplicons were pre-
cipitated by classical ethanol precipitation method (Gismondi et
al., 2012). DNA was sequenced after resuspension in 20 µL of
formamide (100%). Sequencing analysis was performed using
3130 Avant Genetic Analyzer (HITACHI, Applied Biosystems).
Botanical identification of ancient remains was performed ac-
cording to Gismondi et al. (2012). Briefly, high-quality (clearly
Table 1.
Primer sequences.
PRIMER
PAIR NAMESEQUENCE (5’-3 ’)* Tm (˚C) Amplicon (bp)
rbcL F1
rbcL R1
ATGTCACCACAAACAGAGACT
GAATGCTGCCAAGATATCAGT 57.6120
rbcL F2
rbcL R2
ATTATACTCCTGAATACGAAA
TGTCCATGTACCAGTAGAAGA 54130
rbcL F1
rbcL R2
ATGTCACCACAAACAGAGACT
TGTCCATGTACCAGTAGAAGA 52250
rbcL F1
rbcL R3
ATGTCACCACAAACAGAGACT
CTTCTGCTACAAATAAGAATCGAT 57.7670
matK F1
matK R1
GTTCTAGCACAAGAAAGTCGA
CTCAGATTATGATATTATTGA 52.2130
matK F2
matK R2
TGCATATACGCCCAAATCGAT
CCAATTATTCCTCTGATTGGA 54130
matK F1
matK R2
GTTCTAGCACAAGAAAGTCGA
CCAATTATTCCTCTGATTGGA 56.4260
matK F1
matK R3
GTTCTAGCACAAGAAAGTCGA
CCTATCCATCTGGAAATCTTAG 56.8834
trnH-psbA F1
trnH-psbA R1
ATAAAGGAGCAATAACGCCCT
CGAAGTTCCATCTACAAATGG 60120
trnH-psbA F2
trnH-psbA R2
GGTAATGTAACGAATAAAAGT
GAGCAATAAACTCTTTCTTGT 52120
trnH-psbA F1
trnH-psbA R2
ATAAAGGAGCAATAACGCCCT
GAGCAATAAACTCTTTCTTGT 60240
trnH-psbA F3
trnH-psbA R3
CGCGCATGGTGGATTCACAATCC
GTTATGCATGAACGTAATGCTC 55.5525
Primer pair names (F: forward; R: reverse), their sequences (5’-3’), the melting
temperature (˚C) used in PCR amplifications and amplicon lengths in basepairs
(bp). *References: Kress et al., 2005; Kress and Erickson, 2007; Gismondi et al.,
2012.
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A. GISMONDI ET AL.
readable) aDNA regions were aligned and compared with se-
quences registered in scientific databases (GenBank/EMBL/
nucleotide) by Nucleotide Basic Local Alignment Search Tool
algorithm (BLAST, blastn, http://blast.ncbi.nlm.nih.gov/Blast),
with a relaxed E-value cutoff. Resulting species list obtained
for each sample was simplified according to morphological
comparison of ancient remains with putative modern ones (di-
mension, shape and surface), possible geographical distribution,
scientific/historical literature data, maximum values of nucleo-
tide sequence max identity between subject and query and, in
particular, by the intersection of result lists obtained from the
same sample for different barcode regions, as performed in
Gismondi et al. (2012).
Combustion Test and Dry Distillation Assay
Combustion test and dry distillation assay were performed on
fiber samples, according to Goodway (1987) and Quaglierini
(2012). Briefly, in the combustion test, specimens were drawn
up to fire: a rapid combustion and the smell of burnt paper are
typical of vegetal samples, with respect to animal fibers that are
characterized by a slow combustion time and a smell of burnt
hairs. In the dry distillation assay, fibers were placed in a well-
closed flask with a litmus paper, previously dipped in H2Odd.
Then, the flask was put on fire and fumes, released from dis-
solved fibers, permeated the litmus paper that changed its color
(slightly acid pH for samples of flora origin and slightly basic
pH for animal fibers).
Results
Flora ancient remains, found in a sunken amphora in the ar-
chaeological site of Pyrgi (Santa Severa, Rome), were scien-
tifically analyzed in order to taxonomically identify them. To
facilitate this study, samples were named with alphabetic letters
(from A to F).
Sample A
Probably because of time degradation, atmospheric agents or
animal or human action, this sample wasn’t conserved in one
piece. Nevertheless, it appeared as an elongated seed of 0.77 cm
in length (Figure 1(A)). SEM observations showed that seed
coat was wrinkled and characterized by compact and woody
isodiametric cells (Figure 2(A)) (Barthlott, 1981). Tegument
depth constantly measured about 400 µm and it also reported a
loose matrix, due to the presence of collapsed parenchyma cells
(Figure 2(A2)). Morphological analysis couldn’t determine a
taxonomic identification of the specimen; so, the genetic ap-
proach was fundamental. aDNA was extracted from the vegetal
remain and specific genes were amplified (as reported in “Ma-
terials and Methods”). It wasn’t possible to amplify all chosen
barcode regions but, with respect to the other specimens, this
sample showed the best conserved template. Positive PCR pro-
ducts (rbcL F1-R1, rbcL F2-R2, matK F1-R3, trnH-psbA F1-
R1, trnH-psbA F2-R2, trnH-psbA F3-R3) were visualized on
agarose gel, under UV-light, and verified in size, with respect
to the standard molecular weight (MW) (Figure 3). Each am-
plicon was sequenced and compared with the scientific nucleo-
tide databases: this process generated species lists that respec-
tively matched with every analyzed barcode sequence (Supple-
mental Data 1-Sample A). Incompatible species, according
Figure 1.
Archaeological remains have been documented with photos for mor-
phological studies: A) sample A; B) sample B; C) sample C; D) sample
D; E) sample E; F) sample F. Black bars indicate 1 cm.
to the present morphological study, natural geographical distri-
bution and scientific/historical literature data (Liphschitz et al.,
1991; Carrión et al., 2010; Milanesi et al., 2011), were elimi-
nated from the resulting lists. The intersection of these out-
comes allowed us to scientifically reveal the botanical origin of
the sample A: Olea europaea L.
Sample B
In the archaeological amphora twenty-two generally round
seeds, of 0.3 - 0.6 cm in diameter, were found (Figure 1(B)).
The outside of these samples was quite regular though slightly
rough and presenting some little dips (Figure 2(B)). No other
particular characteristics were evidenced by microscopic analy-
sis. The genetic approach was essential for the recognition of
these flora remains: unfortunately, it was possible to amplify
only one region of the maturase K barcode gene (matK F1-R2)
(Figure 4). The relative nucleotide sequence showed a maxi-
mum identity and E-value (89%, 7e-66) with Crataegus genus
species (Supplemental Data 2-Sample B). In particular, accord-
ing to morphological results (here reported and published also
in other works) (Dietsch, 1996; Groningen Institute of Archae-
ology and the Deutsches Archäologisches Institut, 2006), we
concluded that samples B were Crataegus monogyna Jacq.
eeds. s
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A. GISMONDI ET AL.
Open Access 243
Figure 2.
SEM observations of botanical remains: A) sample A surface; A2) sample A tegument deep; B) sample B; C) sample C; C2) particular of sample C;
D) sample D. Enlargement and unit bars are reported in each image.
2(C)). In fact, a lot of prominent protrusions could easily be
individuated: these extroflexions represented the sites where
microsporophylls were attached and held on the pollen cone
central axis. In Figure 2(C2) (inside circles), it was also possi-
ble to distinguish some vascular tissue remains, characterized
by series of tracheids. aDNA was successfully extracted from
Sample C
This specimen, already at first sight, looked like the axis of a
microstrobilus. Its dimensions were 0.72 cm in length and 0.18
cm in width (Figure 1(C)). SEM analysis evidenced a very
irregular surface and structure of the botanical sample (Figure

A. GISMONDI ET AL.
the remain but, maybe because of its excessive degradation,
only one PCR amplification (trnH-psbA barcode gene, region
F3-R3) was obtained (Figure 4). Results, reported in Supple-
mental Data 3-Sample C, confirmed that sample was the fossil-
ized remains of a Pinus sp. pollen cone.
Sample D
Last seed remain, found in the underwater archaeological site,
was clearly an acorn. The pericarp was very reduced and com-
pletely protected by a thick cupule. The whole structure was
rounded, sub-spherical and measured about 0.4 cm (Figure
1(D)). Cupule scales were lanceolated and embricated (Figure
2(D)). Molecular study was performed on the oak nut and the
single obtained amplicon (rbcL F1-R2) was fractionized on
agarose gel (Figure 4). Positive PCR product was analyzed and
matched up with nucleotide sequence databases. Outcomes,
reported in Supplemental Data 4-Sample D, showed high se-
quence identity (95%) of the sample with Quercus genus. In
particular, according to cupule specific morphological features
(Pignatti, 1982; Heinz and Barbaza, 1998), this specimen was
identified as Quercus petraea (Mattuschka) Liebl. fruit.
Sample E
No aDNA trace was detected in sample E. Therefore, for its
taxonomic identification, our research was essentially based on
morphological and microscopic observations. This specimen
presented a filamentous structure (Figure 1(E)). At first, we
considered the possibility that it could be a vegetal fiber (cot-
ton-Gossypium sp. L., linen-Linum usitatissimum L., hemp-
Cannabis sativa L., jute-Corchorus sp. L. or ramie-Boehmeria
nivea L.). Light microscope image (Figure 5(E1)) showed that,
in reality, the sample was made up of several united filaments.
Each of these filaments appeared quite uniform, internally full
and not characterized, on its surface, by the presence of cell
walls (Figures 5(E2), (E3), (E4) and (E5)). According to these
preliminary results, the ancient remain could be neither fossil
cotton nor linen remains: in fact, Gossipium sp. L. product is
Figure 3.
PCR amplifications of sample A aDNA have been
detected by UV-light after migration on agarose gel
1.5%. MW (molecular weight; 100, 500 and 800
base pairs are indicated); lane 1 (rbcL barcode gene,
region F1-R1, 130bp); lane 2 (rbcL barcode gene,
region F2-R2, 130 bp); lane 3 (matK barcode gene,
region F1-R3, 834bp); lane 4 (trnH-psbA barcode
gene, region F1-R1, 120bp); lane 5 (trnH-psbA bar-
code gene, region F2-R2, 120bp); lane 6 (trnH-
psbA barcode gene, region F3-R3, 525bp).
Figure 4.
Positive PCR products visualized on aga-
rose gel 1% by UV-light. MW (Molecular
weight; 200 and 600 base pairs are indi-
cated), lane D (sample D aDNA, rbcL bar-
code gene, region F1-R2, 201bp), lane B
(sample B aDNA, matK barcode gene, re-
gion F1-R2, 200bp); lane C (sample C
aDNA, trnH-psbA barcode gene, region F3-
R3, 525bp).
flat, ribbon-like and helix packaged (Han et al., 1998; Krakh-
malev & Paiziev, 2006) whilst L. usitati ssimum L. fiber is hollow
and characterized by horizontal rings that regularly recur along
the filament (Florian et al., 1990). Hemp, jute and ramie fila-
ments were also observed by optical microscopy (Figures 5(H),
(J) and (R)), in order to discover some similarities between
them and the ancient specimen. Unfortunately, all these fibers
appeared very different with respect to the archaeological sam-
ple. Ramie fiber only presented a remote likeness with the sam-
ple E. So, to clarify this doubt, SEM analysis was performed on
the ancient remain (Figures 6(E1), (E2) and (E3)) and ramie
filaments (Figures 6(R1), (R2) and (R3)). Both fibers meas-
ured about 30 - 35 µm in thickness but, outwardly, they were
very dissimilar: sample E showed a uniform and quite smooth
surface with respect to ramie one that was characterized by
filamentous micro-subunits. All these data suggested that sam-
ple E was not a vegetal fiber. Thus, we have wanted to demon-
strate that it had an animal origin. Apart from the slow burning
time of the sample and the smell of burnt hairs that was pro-
duced during the empiric combustion test, we also observed
that, in the dry distillation assay, litmus paper slightly changing
its color towards basic pH (Figure 7), confirming the animal
nature of the ancient remain. Probably it was raw silk, accord-
ing to Goodway (1987) observations.
Sample F
Last samples were represented by woody fragments (Figure
1(F)). DNA barcoding technique wasn’t applicable, in this case,
because of the absence or too low levels of aDNA. Conse-
quently, only microscopic approach allowed us to obtain some
information about them. Remain sections clearly showed the
Open Access 244

A. GISMONDI ET AL.
Open Access 245
lar and observational approaches, in order to carefully recog-
nize it. In particular, for the genetic characterization, the best
presence of vascular bundles between intrafascicular paren-
chyma (Figure 8(F1)). By the enlargement of these fascicles
(Figures 8(F2) and (F3)), lignified cell walls of xylem tissue
were easily detectable. In particular, as tracheids but not vessel
elements could be distinguished in the section, we concluded
that ancient woods belonged to gymnosperm plants (Minnis,
1987; Yaman, 2011).
Discussion
Ancient DNA (aDNA) is the most important and informative
biological component that scientists can find in archaeological
areas (Gugerli et al., 2005). In fact, its analysis allowed re-
searchers to explain, understand and know a lot of sides of hu-
man past and habitat. The presence of aDNA was also detected
in underwater conditions and sunken ancient structures (Coolen
& Gibson, 2009; Schlumbaum et al., 2012). These data en-
couraged the development of this project whose object was the
taxonomic classification of botanical remains found in the un-
derwater archaeological site of Pyrgi (Santa Marinella, Rome).
Moreover, this study also shot for both promote further the use
of DNA barcode scientific method, only lately applied to ar-
chaeobotany (Gould et al., 2010; Gismondi et al., 2012), and
reassess the application of different microscopic techniques, in
the cases where aDNA was too much damaged or not informa-
tive. In particular, in our previous work (Gismondi et al., 2012),
we demonstrated that morphological observations of macro-
remains, also if well preserved, could sometimes induce to
sample misclassifications. For this fact, the present work wants
to underline the importance of the synergy of different tech-
niques to scientifically identify past plant species. At first, as
genetic investigation would have required specimen destruction,
all flora ancient remains (6 different typologies, named from A
to F) were photographed for macroscopic and metric documen-
tation (Figure 1). Each sample was analyzed, by using molecu-
Figure 5.
Light microscope images. E1, E2, E3, E4 and E5) Sample E at different
enlargement (4X-25X), H) hemp (25X), J) jute (10X); R) ramie (25X).
Mag = 202 X
EHT = 10.00 kV
100 m
Detector = SE1
Date: 30 Nov = 2007
Detector = SE1
Date: 8 Nov 2011
Mag = 1.00 KX
EHT = 10.00 kV
30

m
100 m
Mag = 300 X
EHT = 10.00 kV Mag = 1.00 KX EHT = 10.00 kV
10 m
Detector = SE1
Date: 8 Jan 2009
Detec to r = SE1
Date: 21 Nov = 2011
Mag = 1.00 KX
EHT = 10.00 kV
30 m
Detector = SE1
Date: 8 Nov 2011
Mag = 1.00 KX
EHT = 10.00 kV
20 m
Detector = SE1
Date: 8 Jan 2009
Figure 6.
SEM images of sample E (E1, E2, E3) and ramie (R1, R2, R3). Enlargement and unit bars are reported in each image.

A. GISMONDI ET AL.
Figure 7.
Litmus paper (on the right), used in the dry distil-
lation assay, slightly changing its color towards
basic pH with respect to control litmus paper (on
the left), only imbued with H2Odd.
Figure 8.
Light microscope images of sample F sections. Enlargement: 10× for
F1, 40× for F2 and F3.
plant barcode genes, reported in literature (Kress and Erickson,
2008), were chosen. As aDNA could easily present double
strand breaks, to obtain at least one positive amplification, for
each target gene, we tried to amplify more regions. So, primers
were designed to produce very short (80 - 300 bp) and overlap-
ping or consequent amplicons, according to Schlumbaum et al.
(2008) and Gismondi et al. (2012). However, we also subjected
aDNA to PCR amplifications of longer sequences (>500 bp) in
order to determine if the template hadn’t an excessive deg-
radation level. Optimal preservation conditions of the samples
can represent, in archaeogenetics, a crucial element: undam-
aged aDNAs, usually, are extracted from remains that were
found frozen, desiccated or conserved in oxygen-free environ-
ments (Palmer et al., 2012). Probably because Pyrgi flora re-
mains were preserved in a hermetically sealed amphora, al-
though submerged, no external contamination or excessive phy-
sical and chemical degradation was produced on samples. In
fact, in spite of the adverse environmental situation, specimen
morphological and genetic studies were successfully complet-
ed. In particular, with great surprise and fortunately, aDNA ex-
tracted from A and C samples allow us to amplify even long
nucleotide sequences (Figures 3 line A3 and A6, Figure 4 lane
C), demonstrating a good preservation degree of ancient nucleic
acids. In addition to anoxic conditions, in fact, remains were
maybe highly preserved from the degradation by the presence
in the archaeological vessel of vegetables rich in antioxidant
molecules (Hansson and Foley, 2008). However, the production
of long amplicons starting from ancient or degraded DNA is not
so uncommon (Willerslev & Cooper, 2004; Yoccoz et al.,
2012). Specimen aDNA amplifications (Figures 3 and 4), their
sequencing and matching with nucleotide databases (Supple-
mentary information files), in association with microscopic ana-
lyses (Figure 2), accurately allowed the identification of the
botanical origin of sample A, B, C and D. In particular, for
sample B classification the genetic analysis was essential.
Therefore, aDNA techniques, even if time-consuming and ex-
pensive methods, often represent an accurate and rigorous ap-
proach able to support and balance morphological analysis
lacks. Unfortunately, aDNA wasn’t detectable from E and F an-
cient remains. In these cases, light microscopy (Figures 5 and
8), SEM analysis (Figure 6) and other experimental assays
(Figure 7) were necessary.
Since the archaeological amphora was identified by Enei et
al. (2008) as an ancient replaced of garbage can, vegetal re-
mains, identified and classified in the present research (Olea
europaea L. and Crataegus monogyna Jacq. seeds, a Pinus sp.
pollen cone, a Quercus petraea (Mattuschka) Liebl. acorn, ani-
mal fibers and gymnosperm woody fragments), surely could be
considered as food leftovers or remainders of antique tools.
Therefore, this work increased insights about Central Italy past
human activity and habitat of the 1st Century BC: in fact, it in-
dicated the possible vegetation history and the plant species that
were used, grown or traded by people of that period. On the
other hand, we improved and encouraged further the complemen-
tary application of microscopic techniques and DNA barcoding
to archaeobotany, underlining the innovation of its relevance
also on samples derived from underwater archeological areas.
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Supplemental data files (1, 2, 3 and 4) are available on
demand contacting the corresponding author
Open Access 248