Marine Genomics 24 (2015) 185–196

Contents lists available at ScienceDirect

Marine Genomics

journal homepage: www.elsevier.com/locate/margen

Review

Origin, dynamics, and implications of extracellular DNA pools in

marine sediments
Andrea Torti a,⁎, Mark Alexander Lever b, Bo Barker Jørgensen a
a
Center for Geomicrobiology, Department for Bioscience, Aarhus University, Ny Munkegade 116, Building 1535 DK-8000 Aarhus C, Denmark
b
Institute of Biogeochemistry and Pollutant Dynamics, Department of Environmental Systems Sciences, Swiss Federal Institute of Technology Zurich (ETHZ), Universitätstrasse 16, 8092,
Zürich, Switzerland

a r t i c l e i n f o a b s t r a c t

Article history: In marine sediments, DNA occurs both inside and outside living organisms. DNA not enclosed in living cells may ac-
Received 27 August 2015 count for the largest fraction of total DNA, and include molecules locked within dead cells, organic and inorganic ag-
Accepted 29 August 2015 gregates, adsorbed onto mineral matrices, and viral DNA. This DNA comprises genetic material released in situ from
sediment microbial communities, as well as DNA of pelagic and terrestrial origin deposited to the seafloor. DNA not
Keywords:
enclosed in living cells undermines the assumption of a direct link between the overall DNA pool and the local, cur-
Soluble DNA
Non-soluble DNA
rently living microbial assemblages, in terms of both microbial cell abundance and diversity. At the same time, the
Ancient DNA extracellular DNA may provide an integrated view of the biodiversity and ecological processes occurring on land, in
Fossil DNA marine water columns, and sediments themselves, thereby acting as an archive of genetic information which can be
Microbial diversity used to reconstruct past changes in source environments. In this review, we identify and discuss DNA pools in ma-
Extracellular RNA rine sediments, with special focus on DNA not enclosed in living cells, its origin, dynamics, and ecological and meth-
odological implications. Achievements in deciphering the genetic information held within each DNA pool are
presented along with still-standing challenges and major gaps in current knowledge.
© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Contents

1. DNA pools in sediments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186

1.1. Definitions and properties of extracellular DNA pools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186
1.2. Abundance of DNA not enclosed in living cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
2. Origin of DNA not enclosed in living cells in marine sediments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
2.1. Local production and external input . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
2.2. Active DNA release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
2.3. Passive DNA release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
3. Fates of DNA in the environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
3.1. Natural transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
3.2. Biotic degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
3.3. Abiotic decay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
3.4. Long-term preservation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
4. Disclosing the genetic information of sedimentary DNA pools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
4.1. Separation and comparison of DNA pools: Achievements and challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
4.2. Tracking past biodiversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
4.3. Current limitations of ancient DNA studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
5. RNA in the extracellular environment? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
6. Summary and outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
Conflicts of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194

⁎ Corresponding author.
E-mail addresses: torti@bios.au.dk (A. Torti), mark.lever@usys.ethz.ch (M.A. Lever), bo.barker@bios.au.dk (B.B. Jørgensen).

http://dx.doi.org/10.1016/j.margen.2015.08.007
1874-7787/© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
186 A. Torti et al. / Marine Genomics 24 (2015) 185–196
1. DNA pools in sediments
As the repository of genetic information, deoxyribonucleic acid
(DNA) is inherently associated with all environments on Earth where
life exists. The nucleotide sequence of DNA holds key information
about the identities of organisms, how they function and interact with
each other and their environment, and their evolutionary history. Be-
cause DNA replication and repair are energy-consuming processes
that require sophisticated cellular machineries (Berg et al., 2002), the
genetic information encoded in DNA can only be maintained by living
organisms. Nonetheless, as many other biomolecules (Bianchi and
Canuel, 2011), DNA can persist in the environment after the death of
the source organism. DNA occurs in the necromass (dead biomass)
and the extracellular medium of nearly all of Earth's ecosystems, includ-
ing water columns, soils, and sediments (Dell'Anno and Danovaro,
2005; Nielsen et al., 2007; Pietramellara et al., 2009), the latter
representing the focus of this review.
1.1. Definitions and properties of extracellular DNA pools
DNA occurs in many forms in sediments, which can be grouped to-
gether based on different properties to define distinct DNA pools
(Fig. 1). In the scientific literature, the term extracellular DNA (eDNA)
is often used as a synonym for DNA not enclosed in living cells, as op-
posed to intracellular DNA (iDNA) to indicate DNA belonging to living
organisms. Corinaldesi et al. (2008) broadly defined eDNA as “DNA
that is not associated with living biomass”, thus implying that all DNA
of dead organisms is extracellular, and that intracellular DNA is always
Fig. 1. DNA pools in marine sediments. In this sketch, intracellular DNA and extracellular
DNA are defined simply based on whether they occur inside or outside cells, respectively,
regardless of the physiological status of the cell (alive, dormant, dead). Soluble DNA
consists of dissolved DNA (i.e. fully solubilized in the interstitial water) and molecules
adsorbed onto sediment minerals via electrostatic interaction, which are readily displaced
from the sediment matrix by washing with alkaline, phosphate buffers, whereas non-sol-
uble DNA is released only after harsh physical/chemical lysis treatments. Non-soluble DNA
includes organically/inorganically complexed DNA (comprised of molecules complexed to
insoluble detrital organic inorganic components, or locked inside sediment aggregates),
DNA within structurally intact dead cells, and DNA within living cells). Arrows indicate
that virions may belong to either the non-soluble or the soluble DNA pool, depending
on whether they are attached to particles (as is probably the case for most virions;
Danovaro and Middelboe, 2010), or dispersed in the interstitial water, respectively.
associated with living biomass. A more descriptive definition was
given by Nielsen et al. (2007): “We define extracellular, naked, free, am-
bient, or environmental DNA as: those molecules present in, or released
from, cells in which energy production has permanently ceased,
viral DNA, and DNA secreted from metabolically active cells”. DNA
inside structurally intact dead microbial cells (Boere et al., 2011c;
Manini and Danovaro, 2006), and fossilized plant and animal tissues
(Anderson-Carpenter et al., 2011; Jørgensen et al., 2012; Liepelt et al.,
2006) would not fall into this category. Although enclosed in cells, this
DNA inside dead cells would counterintuitively be classified as eDNA ac-
cording to the definition by Corinaldesi et al. (2008), or be ignored if
DNA pools were only subdivided into eDNA and iDNA pools as in
Nielsen et al. (2007). A similar argument can be made for DNA enclosed
in spores, which are widespread and abundant in marine sediments
(Lomstein et al., 2012). As it is not clear at what point spores lose their
potential to revert to a vegetative lifestyle, it may be difficult to assign
their DNA to either the iDNA or eDNA pool. Incorporation of viral DNA
in the definition of eDNA is also problematic, as viral DNA may occur in-
side cells as prophage (a viral genome integrated in the host's genome),
which may be the main viral proliferation mode in the marine subsur-
face (Engelhardt et al., 2013). Furthermore, the inclusion of extracellu-
lar, ambient, and environmental DNA under the same definition could
be a source of confusion, as ambient and environmental DNA are most
commonly applied to DNA directly extracted from environmental sam-
ples (Taberlet et al., 2012), regardless of whether the DNA is intracellu-
lar vs. extracellular, or associated living biomass vs. necromass.
Throughout this review, the terms eDNA and iDNA will designate DNA
outside and inside structurally intact cells, respectively, regardless of
their physiological status.
The terms “naked” and “free” DNA may also be misleading. It is well
known that DNA can bind to various sediment components, such as
clay, quartz, feldspar and refractory organic molecules (Crecchio and
Stotzky, 1998; Levy-Booth et al., 2007; Lorenz and Wackernagel, 1992;
Nielsen et al., 2006; Siuda and Chrost, 2000). Of these, the interaction
of DNA with inorganic minerals is the best characterized. Adsorption
of DNA onto minerals is rapid, and occurs within minutes on purified
sand and clay (Lorenz and Wackernagel, 1994). At the circumneutral
pH normally found in marine sediments, the interaction is electrostatic,
and relies on inorganic cations (Na+, K+, NH+ 4 , Mg
2+
, Ca2+, Mn2+,
Fe2+/3+, Al3+) to bridge negatively charged surfaces of clay and sand
to the phosphate groups of DNA (Lorenz and Wackernagel, 1987).
Several parameters influence DNA adsorption on minerals, such as the
type of the mineral, the valence and concentration of the bridging
cations, and the pH, and have been extensively reviewed in Lorenz
and Wackernagel (1994) and Levy-Booth et al. (2007). The highest
adsorption capacity (tens of milligrams of DNA per gram of mineral)
is generally observed on clays (Lorenz and Wackernagel, 1994). The
adsorption capacity of sand is typically hundred-fold lower compared
to clay, primarily due to a smaller surface area, and that of silt is expect-
edly intermediate (Levy-Booth et al., 2007). Therefore the mineralogical
composition (i.e. the proportion of clay, silt, and sand) will affect the
amount of mineral-adsorbed DNA (Blum et al., 1997). Once established,
DNA–minerals interactions are tight and stable, and 50 to 95% of the
extracellular DNA seems to exist in this form (Danovaro et al., 2006;
Dell'Anno et al., 2002). While adsorption is often pictured as a simple
monolayer of DNA on top of a mineral grain, evidence exists that
sedimentary organic matter (which includes DNA) can be physically
preserved within clay aggregates made of alternating layers of clay
and organic matter (Salmon et al., 2000). DNA can be easily envisioned
to participate in the formation of such aggregates.
Although less studied, DNA binding to biogenic sediment compo-
nents, such as humic substances and proteins, is known to occur
(Levy-Booth et al., 2007; Nielsen et al., 2006). Humic acids, a heteroge-
neous class of molecules derived from the degradation of various bio-
molecules (Stevenson, 1994), are also capable of binding DNA through
cation bridging (Crecchio and Stotzky, 1998). Proteins are often found
 A. Torti et al. / Marine Genomics 24 (2015) 185–196
in association with DNA. For instance, eukaryotic DNA is tightly
wrapped around histones in vivo (Wolffe, 1998), while prokaryotic, mi-
tochondrial and chloroplast DNA similarly interacts with histone-like
proteins (Caron et al., 1979; Varshavsky et al., 1977). Because proteins
themselves interact with minerals, they can favor DNA adsorption
(Nielsen et al., 2006). Complexation of DNA with reactive iron phases
may be another important interaction. Lalonde et al. (2012) found
that approximately one fourth of the sedimentary organic matter, in-
cluding labile biomolecules such as carbohydrates and proteins, is tight-
ly chelated to iron species, and that this association effectively shields
organic matter from microbial degradation. Because of its known ability
to interact with iron (Ouameur et al., 2005), DNA plausibly participates
in the formation of these chelate.
An additional problem can occur if bulk DNA extractions are per-
formed on complex environmental samples, as the DNA in the final ex-
tract cannot be reliably attributed to any of the original DNA pools. Even
if initial wash steps with DNA-desorbing buffers, e.g. phosphate buffers,
are performed to separate eDNA from iDNA, it is unlikely that they will
extract organically complexed DNA or DNA from inside dead cells. In-
stead DNA pools that were organically complexed or from inside dead
cells are more likely to end up in iDNA extracts, where they might be er-
roneously interpreted to represent living biomass. Thus, until a better
understanding of the sources and turnover of various DNA pools in sed-
iments has been reached, definitions based on operational properties of
different DNA pools might offer valuable insights. Accordingly, Lever
et al. (2015b) subdivided DNA in sediments into two operational
pools: one pool can be solvent-extracted by washing, using high-pH
phosphate buffers in the absence of observed cell lysis. Because this
DNA pool goes into solution without further chemical or physical treat-
ment it was termed soluble DNA (sDNA). sDNA likely consists of DNA
that is reversibly adsorbed onto mineral surfaces, readily displaced in
the presence of phosphate species (e.g. orthophosphate, pyrophos-
phate, metaphosphate, nucleotide triphosphates, etc.) that compete
for the same absorptive sites. By contrast, DNA locked within (living
or dead) cells or sediment-attached viruses, or complexed to insoluble
organic and inorganic matrices and aggregates, which is released only
after cell envelopes, DNA-binding proteins, and other insoluble struc-
tures have been dismantled by harsh physical and/or chemical lysis
agents, belong to the so-called non-soluble DNA pool (nsDNA). It should
be noted that sDNA represents a fraction of the eDNA pool consisting of
adsorbed DNA, DNA dissolved in pore water, and dissolved viruses. By
contrast, nsDNA includes iDNA (living and dead cells), proviruses and
sediment-attached viruses, and organically and inorganically com-
plexed eDNA pools (Fig. 1).
1.2. Abundance of DNA not enclosed in living cells
By comparing the total amount of DNA and the number of microbial
cells hosted in surface oligotrophic sediments from the Eastern Mediter-
ranean Sea, Danovaro et al. (1993, 1999) showed that most (N 90%) of
the sedimentary DNA could not be accounted for by microbial standing
stocks. Part of this DNA is enclosed in DNA viruses, but, even with as
many as 107–1010 units per gram of sediment (Danovaro et al.,
2008a), viral particles seem to represent just a minor fraction of the
cell-free DNA pool (Dell'Anno and Danovaro, 2005). In order to quantify
the fraction of sedimentary DNA not enclosed in living cells, a method
was developed by Dell'Anno et al. (2002) based on the notion that, un-
like DNA locked within cells or viral particles, cell-free DNA should be
degraded into nucleotides by treating the sample with deoxyribonucle-
ases (DNases). This method confirmed that approximately 90% of the
total DNA pool is prone to enzymatic hydrolysis, and therefore truly ex-
tracellular (Dell'Anno et al., 2002; Dell'Anno and Danovaro, 2005). On a
global scale, this fraction corresponds to about 0.30–0.45 gigatons of
DNA in the uppermost 10 cm, and may represent the largest reservoir
of DNA in the world's oceans (Dell'Anno and Danovaro, 2005).
eDNA concentrations have been determined in a variety of marine
187
environments, and values are generally three orders of magnitude
higher in sediments than in marine waters (Table 1). The partitioning
of DNA between living biomass and necromass seems to reflect that of
the overall sedimentary organic pool: using amino acids as a proxy for
microbial biomass, Lomstein et al. (2012) estimated that vegetative
cells only contribute less than 3% of the total carbon pool, the rest be-
longing to microbial necromass.
2. Origin of DNA not enclosed in living cells in marine sediments
2.1. Local production and external input
The accumulation of DNA not enclosed in living cells in marine sed-
iments results from a number of different processes, outlined in Fig. 2.
On one hand, sediments host abundant microbial populations
(Kallmeyer et al., 2012; Whitman et al., 1998) that represent the source
of the autochthonous (i.e. of local origin) fraction of DNA not enclosed in
living cells. Additionally, an allochthonous (i.e. of foreign origin) fraction
reaches the seafloor as part of the sinking biogenic detritus of marine
and terrestrial origin. Sedimentation of DNA released in the water col-
umn requires it to be associated with particles that are large enough
to sink to the seafloor (Herndl and Reinthaler, 2013), such as marine
snow, fecal pellets and massive falls of phytoplankton material after sur-
face blooms, which may agglomerate particles and enable them to sink
rapidly. DNA of terrestrial origin can be conveyed to the marine com-
partment by rivers and winds. It has been estimated that, on a global
scale, rivers discharge 860–14,500 tons of eDNA per year into the oceans
(Overballe-Petersen et al., 2013). The downward flux of particulate
eDNA has been estimated to deposit up to 10 and 550 mg DNA m− 3
year−1 in open-ocean and continental margin sediments, respectively
(Dell'Anno et al., 2005; Dell'Anno et al., 1999). As similar estimates of
local production are currently missing, it remains unknown whether
the sedimentary necromass DNA is mainly comprised of DNA of local
or foreign origin.
2.2. Active DNA release
DNA not enclosed in living cells may derive from several different
pathways of both active release from physiologically active cells and
passive release from moribund or dead cells (Fig. 2). Most bacteria ex-
amined to date secrete naked DNA during growth in vitro (Battista,
1997; Dillard and Seifert, 2001; Hamilton et al., 2005; Lorenz et al.,
1991; Lorenz and Wackernagel, 1994; Matsui et al., 2003; Paget and
Simonet, 1994; Thomas and Nielsen, 2005; Yin and Stotzky, 1997;
Zafra et al., 2012). An increasing number of environmental microorgan-
isms are being found to release DNA within membrane vesicles, such
as the marine cyanobacterium Prochlorococcus (Biller et al., 2014),
Pseudomonas aeruginosa (Renelli et al., 2004), and the hyperthermo-
philic archaea Thermococcus and Pyrococcus (Soler et al., 2008). DNA
secreted by some strains is known to fulfill a structural function by sta-
bilizing and strengthening biofilms (Das et al., 2010; Fröls et al., 2012;
Steinberger and Holden, 2005; Whitchurch et al., 2002).
2.3. Passive DNA release
Passive DNA release from prokaryotes occurs in conjunction with
cell death following viral attack, grazing, or exposure to cytotoxic
agents, which often lead to cell rupture and release of the cytoplasmic
content, including DNA (Levy-Booth et al., 2007; Palmen and
Hellingwerf, 1995, 1997). In marine habitats, viral infection and protis-
tan grazing are the leading causes of prokaryotic mortality (Pernthaler,
2005). Destruction of the infected cell and its membrane, followed by
the release of the cytoplasmic content, is the final step in the viral lytic
cycle, and serves the purpose of liberating the new viral generation
into the environment. Phagotrophic protists are known to release ex-
cess nutrients from ingested bacteria (Nagata and Kirchman, 1991;
188 A. Torti et al. / Marine Genomics 24 (2015) 185–196

Table 1
Quantities of eDNA in various aquatic environments. Expanded and updated from Lorenz and Wackernagel (1994).

Sampling location DNA poola DNA concentrationb Reference(s)

Marine water
Estuarine Dissolved DNA 9.4–11.6 Paul et al., 1989; Paul et al., 1987
” 6.0–44.0 DeFlaun et al., 1987
” 10.0–19.0 DeFlaun et al., 1987
Coastal ” 5.0–15.0 DeFlaun et al., 1986
” 2.0–7.0 Turk et al., 1992
” 5.1 Dell'Anno and Corinaldesi, 2004
Offshore ” 0.2–10.5 DeFlaun et al., 1986; Paul et al., 1989
” 0.2–1.9 DeFlaun et al., 1987
” 17.1 Turk et al., 1992
Freshwater
Oligotrophic ” 0.5–3.2 DeFlaun et al., 1986; Paul et al., 1989
Eutrophic ” 1.1–25.6 DeFlaun et al., 1986; Paul et al., 1989
Swamp ” 7.8 DeFlaun et al., 1986
Sediments
Lacustrine sDNA (nsDNA) 1.0 (8.0) Ogram et al., 1987
Coastal marine Nuclease-digestible DNA 0.5–3.3 Corinaldesi et al., 2007b
” 3.9–9.8 Corinaldesi et al., 2007a
” 8.6–35.0 Dell'Anno and Corinaldesi, 2004
sDNA (nsDNA) 0.626 (0.035) Alawi et al., 2014
sDNA (nsDNA) 0.084–5.960 (0.088–4.496) Lever et al., 2015b
Continental margin Nuclease-digestible DNA 14.6 Dell'Anno et al., 2005
” 22.3 Dell'Anno et al., 2005
sDNA (nsDNA) 0.056 (0.010) Alawi et al., 2014
Deep sea Nuclease-digestible DNA 4.6 Dell'Anno et al., 2005
” 18.4–21.2 Corinaldesi et al., 2007a
sDNA (nsDNA) 0.023 (0.0005) Alawi et al., 2014
Deep-sea (anoxic) Nuclease-digestible DNA 6.7–22.6 Corinaldesi et al., 2007b
a
Dissolved DNA is isolated from water samples by pre-filtration through a 0.2 μm-filter, precipitated in trichloroacetic acid or ethanol, and quantified in a fluorescence or radioactivity
assay. Nuclease digestible DNA is extracted and quantified by the DNase digestion method described in Dell'Anno et al. (2002), while soluble DNA (sDNA) is extracted in alkaline
phosphate buffers. For definitions of sDNA and nsDNA see Lever et al. (2015b) and Section 1.1.
b
Expressed as μg L−1 for water column samples and μg (g wet sediment)−1 for sediment samples. Non-soluble DNA concentrations, where available, are given in parentheses.

Sherr et al., 1983; Taylor and Lean, 1981), including most of the acquired
DNA (Turk et al., 1992). This is thought to occur because, compared to
eukaryotes, bacteria have a higher phosphorous to biomass ratio
(Simon and Azam, 1989), and thus supply their predators with more
phosphorous than is needed for growth. Viral infection and protistan
grazing are acknowledged sources of mortality in benthic ecosystems
as well (Fischer et al., 2006). As shown by Danovaro et al. (2008b),
viruses account for 16% of prokaryotic mortality in surface coastal sedi-
ments. A study by Corinaldesi et al. (2007b) found that the viral infec-
tion contributed to 2–11% of the total eDNA pool in Eastern
Mediterranean deep hypersaline anoxic sediments. However, virus-
mediated eDNA release may not just be restricted to active surface
sediments, as evidence exists for ongoing virus production in deep
sub-surface sediments (Engelhardt et al., 2014).
DNA from fauna and flora is also detected in aquatic environments in
particulate or dissolved form, potentially originating from feces, mu-
cous, gametes, shed cells and tissues, and carcasses (Pilliod et al., 2013).
3. Fates of DNA in the environment
Upon cell death or release in the extracellular environment, DNA
molecules can undergo different processes, which include natural trans-
formation, biological degradation, long-term preservation, and abiotic
decay. These processes can all occur in sediments and are depicted in
Fig. 2.
3.1. Natural transformation
During genetic transformation, extracellular DNA is internalized by
naturally competent microbial cells (i.e. cells capable of taking up free
DNA molecules from the environment) and incorporated into their
genome. Evidence exists that transformation does occur in nature
(Lorenz and Wackernagel, 1994), but the actual rate and evolutionary
impact on environmental microbial communities remain debated
(Overballe‐Petersen and Willerslev, 2014). Earlier investigations found
that efficient uptake of DNA only occurs with kilobase-long molecules,
which may not persist for long in the environment (Nielsen et al.,
2007). This paradigm changed when Overballe-Petersen et al. (2013)
reported the transformation of the soil bacterium Acinetobacter baylyi
with DNA molecules as short as 20 bp. Their study also showed that
A. baylyi can incorporate DNA molecules bearing damages such as abasic
sites, cross-links, and miscoding lesions (see Section 3.3), which are
common in most environments, in addition to DNA from extinct and
unrelated organisms, such as DNA fragments from a 43,000-year old
mammoth bone. Such DNA molecules become incorporated in the
host's genome by simply acting as primers for the synthesis of the
Okazaki fragments during genome replication (Overballe-Petersen
et al., 2013). Because this phenomenon does not require specialized
protein machineries, it is likely widespread in nature, at least among
naturally competent bacteria. A comparative genomic analysis of repre-
sentative non-competent and naturally competent bacterial species in-
deed revealed the impact of natural transformation by fragmented and
damaged DNA in shaping the genomes of the latter group (Overballe-
Petersen et al., 2013). These findings suggest that eDNA potentially
contributes to microbial evolution through natural transformation irre-
spective of its source and structural integrity. The actual extent of gene
flow across microbial cells via eDNA transformation in marine sediments
remains, to our knowledge, completely unaddressed.
3.2. Biotic degradation
Biogeochemical cycling in sediments is largely driven by the decom-
position of detrital organic matter by organotrophic microorganisms
(Arnosti, 2014). Since microbial cells can take up only small molecules
through the cell membrane, they produce enzymes to break down
large organic molecules extracellularly (Arnosti, 2011; Azam, 1998;
Hoppe, 1991). Upon release in the extracellular environment, DNA be-
comes susceptible to extracellular and cell-associated DNases, which
 A. Torti et al. / Marine Genomics 24 (2015) 185–196
Fig. 2. Origin and dynamics of DNA not enclosed in living cells in marine environments. Along with microbial cells, multicellular organisms can passively release DNA. Notice how processes
contributing to the accumulation of DNA not enclosed in living cells in sediments do not only occur within sediments, but also in the overlying water column and surrounding lands.
probably are the most immediate cause of DNA degradation. These en-
zymes are ubiquitous in most environments, including soil, marine wa-
ters, and sediments (Blum et al., 1997; Corinaldesi et al., 2008; Paul
et al., 1989), and catalyze the hydrolysis of the phosphodiester bond be-
tween the phosphate and the deoxyribose moieties of DNA (Fig. 3b).
Nucleotides produced by DNases can then be internalized by microbial
cells and incorporated into newly-synthetized nucleic acids, or further
broken down to provide carbon, nitrogen, and phosphorous
(Jorgensen and Jacobsen, 1996; Jørgensen et al., 1993; Kroer et al.,
1994; Turk et al., 1992). With an elemental composition comprising
40% C, 20% N, and 10% P, DNA represents a valuable source of nutrients
for microbial metabolism. It has been estimated that DNA supplies
benthic heterotrophic communities with 4%, 7%, and 47% of their daily
C, N, and P demand in deep-sea sediments, while these proportions
are 2%, 4%, and 20% in coastal sediments (Corinaldesi et al., 2007a;
Dell'Anno and Danovaro, 2005).
Numerous isolates can grow in laboratory cultures in the presence of
DNA as the sole, or primary, energy substrate. Those include Escherichia
coli (Finkel and Kolter, 2001; Palchevskiy and Finkel, 2006),
P. aeruginosa (Mulcahy et al., 2010), members of the Bacteroidetes,
Alpha- and Gamma-proteobacteria isolated from coastal waters
(Lennon, 2007), several Shewanella species (Pinchuk et al., 2008), and
the halophilic archaeon Haloferax volcanii (Chimileski et al., 2014). No
culture-independent studies have shown how common this metabolic
trait is in the prokaryotic domain besides the species identified in
these studies. However, the biochemical pathways that allow them to
uptake and metabolize DNA have homologs in the genomes of many
other bacterial and archaeal species (Chimileski et al., 2014;
Palchevskiy and Finkel, 2006). The use of DNA as an energy source
189
might therefore be a widespread trait. Not surprisingly, calculated turn-
over times for dissolved DNA in marine waters are fast, and range from
6.5 to 25 h (Paul et al., 1987, 1989). By contrast, turnover times of bio-
available DNA (accessible to nucleases) range from 29 to 93 days in sur-
ficial sediments (Dell'Anno and Corinaldesi, 2004). This difference is
thought to simply reflect higher eDNA concentrations in sediments
compared to seawater.
3.3. Abiotic decay
Upon cell death, DNA repair mechanisms cease to be active, and DNA
will start accumulating lesions inflicted by a number of abiotic (physical
and chemical) factors. These factors promote DNA decay by acting on
the structure of both the individual bases and the overall DNA molecule
(Fig. 3).
Under hydrated conditions, DNA is especially prone to hydrolysis,
oxidation, and UV-mediated damages (Hebsgaard et al., 2005; Lindahl,
1993; Willerslev and Cooper, 2005). Lack of light and free oxygen prob-
ably affords good protection against oxidation and irradiation in most
sediments, leaving hydrolysis as the potential main DNA decay route
in sediments. Hydrolysis is responsible for the loss of purine bases
(depurination, Fig. 3a) and cleavage of the phosphodiester bond
(Fig. 3b), both resulting in single-strand breaks. Depurination rates
vary with temperature according to the Arrhenius equation, so that a
decrease in temperature produces an exponential decrease in the
depurination rates. Approximately, rates decrease by an order of magni-
tude every 16 °C decrease (Lever et al., 2015a), and have been experi-
mentally determined in vitro by Lindahl and Nyberg (1972). Using
these rates, the survival of DNA fragments of a certain length and initial
190 A. Torti et al. / Marine Genomics 24 (2015) 185–196

Fig. 3. Pathways of common DNA degradation processes. a) Loss of a guanine base via depurination, generating an abasic site, followed by strand break via beta elimination. The detailed
mechanism of beta elimination is explained in Doetsch and Cunningham (1990). Depurination occurs at a slightly faster rate for G than for A (Lindahl and Nyberg, 1972), while
depyrimidazion rate (loss of T or C bases) is only 5% of the depurination rate (Lindahl, 1993). During DNA synthesis, abasic sites can either act as blocking lesions, i.e. prevent DNA poly-
merases from incorporating new nucleotides during PCR, or miscoding lesions, i.e. cause the incorporation of a wrong nucleotide. b) Hydrolysis of the phosphodiester bond, resulting in a
single-strand break, which acts as a blocking lesion. The reaction is the same for abiotic and nuclease-catalyzed hydrolyses. c) Hydrolytic deamination of cytosine to uracil. Other deam-
ination reactions involve adenine, guanine and the naturally occurring, modified base 5-methyl-cytosine, yielding hypoxanthine, xanthine and thymine, respectively (not depicted). De-
aminated bases have different pairing affinities than their precursors (uracil pairs to adenine, hypoxanthine to cytosine, xanthine to thymine, and thymine to adenine), thereby causing
incorporation of wrong nucleotides during PCR. d) UV-induced formation of a thymine photodimer. This reaction can involve any couple of adjacent pyrimidines. e) and f) are oxidation
reactions converting guanosine to 8-oxo-guanine and cytosine to 5-hydroxy-hydantoin, respectively. A 5-methyl-5-hydroxy-hydantoin can form from thymine oxidation (not depicted).
By pairing up with adenine instead of cytosine, 8-oxo-guanines can introduce mutations during PCR, while hydantoins can block DNA polymerases, as well as cause formation of chimeric
PCR products. g) Intermolecular damages. Interstrand crosslinks (ICLs) can form between the two strands of the same double helix or two different helices. By preventing full separation of
DNA strands, ICLs hinder PCR amplification. Bulky adducts with a variety of molecules (e.g. with a protein in the figure) can also negatively affect PCR.

concentration can be estimated. Pääbo and Wilson (1991) predicted
that, starting with 1012 copies of 800-bp long, fully hydrated DNA mol-
ecules, no fragments will survive undamaged after 5,000 years at 15 °C
and pH 7. Under the same conditions, depurination of an equal number
of 150-bp long molecules would require 26,700 years (Golenberg et al.,
1991). The rate of depurination decreases with increasing pH for pH
values between approximately 4.5 and 7.5. Additionally, depurination
rates decrease with increasing ionic strength. A 10 mM NaCl concentra-
tion is sufficient to produce a sevenfold rate decrease at 45 °C and pH 5
(Lindahl and Nyberg, 1972).
Deamination of nitrogen bases is another well-known hydrolytic
process. Deamination converts adenine and guanine to hypoxanthine
and xanthine, respectively. However, cytosine and its naturally-
occurring analog 5-methyl-cytosine are the main targets for this reac-
tion (Lindahl, 1993; Shapiro, 1982). Deamination of cytosine yields
uracil (Fig. 3c), and deamination of 5-methyl-cytosine yields thymine.
Similarly to depurination, cytosine deamination rates have been mea-
sured in vitro, and increase with increasing temperature and under acid-
ic conditions (Ehrlich et al., 1990; Frederico et al., 1990; Lindahl, 1979;
Lindahl and Nyberg, 1974). In single-stranded DNA, cytosine deamina-
tion and depurination proceed at similar rates. In such DNA, the half-
life of cytosine residues is about 200 years at 37 °C and pH 7.4. However,
the double helix seems to effectively protect cytosines from deamina-
tion, as in double-stranded DNA rates drop to 0.5–0.7% of those in
single-stranded DNA. Deamination rates of adenine are lower (2–3%)
than those of cytosine, and similar to those of guanine (Karran and
Lindahl, 1980). In the dark and anoxic conditions prevailing in many
types of sediments, oxidation and irradiation are unlikely to inflict ex-
tensive damage to DNA. However, it is possible that DNA molecules re-
leased in oxic and/or sunlit environments accumulate oxidative and UV-
 A. Torti et al. / Marine Genomics 24 (2015) 185–196
induced lesions before being deposited and buried in the sediments. UV
radiation is responsible for the formation of pyrimidine dimers (Fig. 3d).
Oxidative damages include the formation of 8-hydroxyguanine from
guanine (Fig. 3e), and of hydantoins from pyrimidines (Fig. 3f). Finally,
interstrand crosslinks (ICLs) can form between the two strands of the
same double helix or two different helices, while adducts can form
upon covalent bonding of DNA with other biomolecules, such as pro-
teins (Fig. 3 g).
As explained in Section 4.3, all these types of damages variably affect
our ability to study ancient DNA sequences.
3.4. Long-term preservation
The co-occurrence, in marine sediments, of active extracellular DN-
ases along with large quantities of their substrate eDNA (Corinaldesi
et al., 2008; Dell'Anno and Corinaldesi, 2004), still represents a partially
unsolved conundrum, which cannot be explained only on the basis of a
higher rate of DNA release in the extracellular medium over the degrada-
tion processes. Indeed, eDNA concentrations are high even in the absence
of continuous eDNA supply from the water column (e.g. in sub-surface
sediments) and in sediments with very low in situ eDNA production
(Corinaldesi et al., 2007a, 2008; Dell'Anno et al., 2005). One plausible ex-
planation is DNA protection from nucleases upon adsorption onto the
sediment matrix. Compared to dissolved DNA, DNA adsorbed onto mont-
morillonitic clay requires 10-fold higher DNase concentrations to achieve
the same extent of degradation (Khanna and Stotzky, 1992). DNA
adsorbed to sand is even more refractory, requiring 100 to 1,000 times
more DNase (Romanowski et al., 1991). The molecular basis for protec-
tion of adsorbed DNA is not completely understood. It has been suggested
that adsorption onto minerals decreases DNA accessibility to nucleases
(Khanna and Stotzky, 1992; Lorenz and Wackernagel, 1987, 1992; Paget
et al., 1992; Romanowski et al., 1991). However, nucleases themselves
adsorb to sand and clay (Khanna and Stotzky, 1992), and may thereby be-
come inhibited or inactive, similar to other clay-adsorbed enzymes
(Sarkar et al., 1989). Binding to biogenic sediment components, such as
humic acids and proteins, may also enhance DNA resistance against nu-
cleases (Nielsen et al., 2006). Kelman and Moran (1996) proposed the as-
sociation of eukaryotic DNA with histones, and of prokaryotic,
mitochondrial and plastid DNA with histone-like proteins to play a crucial
role in shielding DNA from nucleases. Inclusion in dead cells may also ren-
der DNA inaccessible to extracellular nucleases (Novitsky, 1986). Other
mechanisms of organic matter preservation in sediments that are
known to shield labile biomolecules (e.g. proteins, carbohydrates, and
pigments) from microbial degradation, such as inclusion in iron chelates
(Eglinton, 2012; Lalonde et al., 2012), clay aggregates (Salmon et al.,
2000), sulfurization (i.e. the formation of organosulfur compounds upon
reaction of labile organic matter with sulfide and polysulfides; Damsté
et al., 1989; Moodley et al., 2005; Van Kaam-Peters et al., 1998), and inter-
action with exopolymeric substances (Pacton et al., 2007) may provide
additional mechanisms of DNA preservation in sediments.
As discussed in Section 3.3, physical and chemical parameters can
greatly influence rates of abiotic DNA decay and, when favorable,
stretch DNA survival over millennial timescales. Not surprisingly, the
oldest authenticated DNA sequences were derived from frozen environ-
ments, such as permafrost and ice cores (Willerslev et al., 2004a,b), and
from marine sediments deposited under anoxic conditions (Boere et al.,
2011a). Thanks to its preservation potential in sediments, DNA
represents a powerful proxy for investigating the biodiversity of
paleoenvironments, as discussed in the following section.
4. Disclosing the genetic information of sedimentary DNA pools
Life at and below the seafloor comprises a vast diversity of microbial
species. These organisms decisively contribute to global living biomass
and biogeochemical cycles (Parkes et al., 2014), yet their identity and
mode of life remain elusive (Jørgensen and Boetius, 2007). Isolation
191
and cultivation-based approaches have limited applicability, as micro-
organisms that are readily culturable on common laboratory media
comprise a minor (0.1–1%) and unrepresentative fraction of the total
microbial diversity (Staley and Konopka, 1985). Molecular biological
techniques such as environmental nucleic acid extraction, PCR amplifi-
cation and sequencing, have made it possible to identify microorgan-
isms through molecular phylogeny (especially of the small subunit
ribosomal RNA genes), thus bypassing the need for prior cultivation
and providing a more realistic picture of the diversity in the microbial
realm (Head et al., 1998; Lauber et al., 2009; Roesch et al., 2007; Sogin
et al., 2006). DNA is most commonly extracted from sediments via
direct lysis, which involves chemical and/or physical disruption of mi-
crobial cells without first separating them from the sedimentary matrix.
Such methods are fast and ensure DNA extraction from most cells in the
population (Moré et al., 1994). Inevitably, though, DNA occurring inside
and outside living cells will be extracted together. This undermines the
assumption of a direct relationship between the total extracted DNA
and the local, currently living microbial assemblages, in terms of both
cell abundance and diversity (Frostegård et al., 1999; Luna et al., 2006).
4.1. Separation and comparison of DNA pools: Achievements and challenges
In an attempt to obtain better insights into the phylogenetic com-
positions of different sedimentary DNA pools, procedures for sepa-
rate extraction of DNA pools have been developed by Ogram et al.
(1987), Corinaldesi et al. (2005), Alawi et al. (2014), and Lever
et al. (2015b). All rely on desorbing DNA adsorbed onto the sediment
matrix by treating the sample with slightly alkaline (pH 8–10)
phosphate-based buffers, followed by centrifugation to separate
the solid phase, which includes DNA in living and dead cells or com-
plexed with particles from the aqueous phase containing this
desorbed DNA. The rationale behind this strategy is that DNA adsorp-
tion decreases in the presence of phosphate, due to competition be-
tween phosphate ions and the phosphate groups in the sugar-
phosphate backbone of DNA (Saeki et al., 2010). As found by Lever
et al (2015b), the amount of added phosphate and the pH of the
phosphate buffer are crucial for maximizing DNA desorption. Cell
lysis during the initial desorption step, which would lead to contam-
ination of the initially desorbed DNA with DNA from living cells,
should be ruled out. This can be done by comparing the number of
cells obtained by direct cell count in samples treated for DNA desorp-
tion and in untreated controls (Lever et al., 2015b). It is worth noting
that phosphate treatments have never been shown to efficiently ex-
tract DNA locked within structurally intact dead cells, microfossils, or
aggregates of particulate matter. Expectedly, extraction of DNA from
those structures may only be accomplished by harsh treatments that
living cells would not withstand. Therefore, despite being generally
stated that phosphate-assisted desorption can achieve separation
of iDNA from eDNA, or even DNA in living biomass from DNA in
necromass, this method probably only allows for separation of solu-
ble and non-soluble DNA pools, as defined in Section 1.1 and Fig. 1.
Using a DNA desorption method based on phosphate buffer and
SDS, Corinaldesi and colleagues showed that sDNA isolated from a
variety of coastal and deep-sea sedimentary settings contains
amplifiable 16S and 18S rRNA gene sequences, though in lower
abundance than in the nsDNA pool (Corinaldesi et al., 2008, 2011).
Based on qPCR quantification of 16S rRNA genes in a wide spectrum
of sediment types, Lever et al. (2015a,b) determined the contribu-
tion of sDNA to the total DNA pool to vary considerably (10–83%).
Moreover, analysis of prokaryotic 16S rRNA gene sequences within
sDNA and nsDNA recovered from surficial oxic and anoxic Mediter-
ranean Sea sediments found only partially overlap between the two
DNA pools, indicating that sDNA may indeed comprise sequences be-
longing to prokaryotes not present in the sediments as living cells
(Corinaldesi et al., 2014).
192 A. Torti et al. / Marine Genomics 24 (2015) 185–196
4.2. Tracking past biodiversity
Although it is still not possible to treat sediment samples so as to
fully separate DNA in living biomass from DNA in necromass, the latter
is sometimes easily recognizable because the source organism is clearly
not adapted to living beneath the seafloor, but is instead indigenous to
pelagic or even terrestrial habitats. This genetic material, often referred
to as ‘ancient’ or ‘fossil’ DNA, is deposited to the seafloor as part of the
sinking organic matter, and persists here, as it gradually becomes bur-
ied, thanks to the preservation mechanisms discussed in Section 3.4.
This DNA thus represents a genetic archive of past biodiversity of the
overlying waters and surrounding lands, which is currently being
explored for its potential in paleoenvironmental sciences.
The identification and enumeration of microscopic biological
remains in sediments, such as fossilized shells of protists (e.g. dia-
toms, dinoflagellates, foraminifera, radiolarians), pollen and parts
of plants and animals is a standard paleoecological approach, rou-
tinely implemented in scientific drilling of marine sediments. How-
ever, the identification of microfossils has two major shortcomings,
namely dissolution processes, which sometimes results in poor pres-
ervation of calcareous and siliceous shells, and the fact that the ma-
jority of species do not leave fossil cellular diagnostic features
(Dale, 1983; Lecroq et al., 2011; Lejzerowicz et al., 2013), thus escap-
ing identification in the sedimentary record. Molecular fossils, such
as lipids and pigments, offer the chance to extend paleontological
and paleoenvironmental studies to organisms whose exoskeletons
fail to fossilize (Briggs and Summons, 2014), and in some cases can
be detected billions of years after deposition (Brocks and Banfield,
2009; Brocks et al., 2005; Kuypers et al., 2001; Summons and
Powell, 1986). However, because most lipid biomarkers are charac-
teristic of broad groups of organisms (e.g. terrestrial plants, algae,
bacteria, archaea) rather than single species, the taxonomic resolu-
tion and specificity of lipid-based stratigraphy is limited (Coolen
and Gibson, 2009).
In comparison, DNA is generally thought to be relatively short-lived:
ancient DNA has not yet been shown to survive in unfrozen sediments
beyond the late Pleistocene era, i.e. beyond the past few hundred thou-
sand years (Boere et al., 2011a,b,c). On the other hand, its source speci-
ficity is unparalleled by any other molecular fossil, with potential
taxonomic resolution to the species level (Edvardsen et al., 2000;
Sogin et al., 1986). Once presumed to be efficiently preserved only in an-
oxic depositional environments due to slower oxidation and microbial
degradation (Coolen et al., 2006; Coolen and Overmann, 1998;
Corinaldesi et al., 2008), sedimentary ancient DNA was thought to be
restricted to anoxic aquatic settings. Yet, ancient DNA is now unambig-
uously being reported from sediments deposited under fully oxic condi-
tions (Lejzerowicz et al., 2013; Coolen et al., 2013). These studies
suggest that planktonic DNA may be much more widely preserved in
Holocene and Pleistocene sediments than previously thought.
For example, ancient DNA has been used to study haptophyte and
dinoflagellate successions in Arctic, Antarctic, Mediterranean and
Black Sea sediments (Boere et al., 2009, 2011b; Coolen et al., 2006;
D'Andrea et al., 2006). Sediment cores from water depths of 5,000 m
in the Atlantic Ocean off Brazil (Lejzerowicz et al., 2013) and 900 m in
the Black Sea (Coolen et al., 2013) yielded ancient DNA sequences
from dinoflagellates, copepods, coccoliths and foraminifera. Based on
fossil findings and DNA, extinctions and colonization of these communi-
ties were reconstructed over the past 14,000 years. In addition to
informing about past environmental conditions, sedimentary ancient
DNA can also give insights about how microbial populations
(co)evolved over millennial timescales and responded to environmen-
tal conditions. For example, genetic signatures of the photosynthetic
haptophyte, Emiliania huxleyi, and its coccolithoviruses retrieved from
sediment cores of the Black Sea revealed changes in host and viral pop-
ulation structures during ca. 7,000 years of Holocene history, and
showed that such changes coincided with shifts in salinity and nutrient
availability (Coolen, 2011). DNA from lacustrine sediments has also
proved useful for tracking human activities and landscape changes in
proximity to an alpine lake throughout the Anthropocene (Giguet-
Covex et al., 2014), and for identifying changes in photosynthetic plank-
ton communities in a Tibetan lake over the last ca. 3,000 years (Hou
et al., 2014).
4.3. Current limitations of ancient DNA studies
The application of ancient DNA for paleoenvironmental studies is
not exempted from potential biases that must be taken into account
when designing ancient DNA studies and interpreting their results.
Most limitations of ancient DNA discussed here, however, are known
to affect reconstructions based on “traditional” proxies, such as molecu-
lar biomarkers and microfossils, thus highlighting the importance of
integrating results from multiple paleonvironmental proxies.
A potential issue is represented by differential preservation of DNA
depending on the source organism. All comparative analyses of ancient
DNA, fossil lipids and microfossils performed so far indeed showed
that the source species can affect the level of DNA preservation
(Boere et al., 2011c; Jørgensen et al., 2012; Lejzerowicz et al.,
2013; Parducci et al., 2015). In a recent study by Capo et al. (2015)
on limnic sediments, 71% of planktonic unicellular eukaryotic taxa de-
tected in the water column via 18S rRNA gene survey were also found
in the DNA from the underlying sediment down to 30 cm below the
lake floor, along with evidence that such DNA did not derive from
active cells. While providing evidence that sediments can archive and
preserve the largest fraction of the planktonic protistan diversity, this
study also found that some important planktonic taxa (such as
Cryptophyta and Haptophyta) were poorly represented in the sedimen-
tary DNA record.
Vertical migration of DNA across sediment strata (leaching),
redeposition from older deposits, and reworking by post-depositional
mixing may pose additional complications. These processes violate the
assumption that ancient DNA sequences were lain down together
with the host deposit, and may therefore confound ancient DNA-
based interpretations. Leaching has mainly been observed in coarse-
textured or unsaturated deposits that allow fluid advection across strata
(Andersen et al., 2012; Haile et al., 2007), and is generally regarded as a
minor issue in compact, permanently water-saturated sediments
(Giguet-Covex et al., 2014). Sediment biostratigraphy based on any
paleoenvironmental proxy can be obscured by redeposition and
reworking, however, and ancient DNA makes no exception (Arnold
et al., 2011). To ensure reliable results, ancient DNA studies should al-
ways be accompanied by a thorough investigation of the stratigraphy
and depositional history of the site.
Ancient DNA studies inevitably rely on PCR and/or sequencing, and
DNA damages (see Section 3.3) can negatively affect the applicability
and the outcome of these techniques. Damages that alter the base-
pairing properties of individual bases, called miscoding lesions, cause
DNA polymerases to introduce mutations (and therefore sequencing er-
rors) by incorporating wrong nucleotides opposite modified bases.
Deamination is a well-known example of miscoding lesion, as
deaminated bases do not pair up with the same bases as their precur-
sors. For example, deamination of cytosine produces uracil, which
pairs up with adenine instead of guanine, thereby resulting in C–T tran-
sitions. This may be one of the major sources of miscoding lesions in an-
cient DNA (Briggs et al., 2007; Brotherton et al., 2007; Hofreiter et al.,
2001). Miscoding lesions can also arise from base oxidation. The
oxidation product of guanine, 8-hydroxyguanine, preferentially pairs
up adenine rather than cytosine, thus generating transversion muta-
tions (Kasai and Nishimura, 1984; Shibutani et al., 1991). Damages
that block DNA polymerase progression during PCR, therefore
preventing amplification of the template sequence, are called blocking
lesions. Hydantoins, pyrimidine dimers, double strand breaks, ICLs and
bulky adduct all fall into this category (Chan et al., 1985; Wellinger
 A. Torti et al. / Marine Genomics 24 (2015) 185–196
and Thoma, 1996; Willerslev and Cooper, 2005). At abasic sites (formed
upon loss of purines, see reaction in Fig. 3a), DNA polymerases can
either stop polymerizing, insert an adenine, or introduce a one-
nucleotide deletion (Cuniasse et al., 1990; Lindahl, 1993; Sikorsky
et al., 2007).
Patterns and rates of ancient DNA damage has been extensively
characterized in situ for certain environmental matrices, such as in ar-
chaeological findings, permafrost and ice cores (Allentoft et al., 2012;
Hansen et al., 2006; Mitchell et al., 2005), which have helped
researchers deal with the issue, including designing strategies to
recover ancient DNA molecules, such as treatments with uracil-DNA-
glycosylase to remove uracil from deaminated cytosines (Willerslev
and Cooper, 2005), and N-phenacylthiazolim bromide to remove ICLs
(Poinar et al., 1998). In contrast, the study of DNA decay routes and
kinetics in marine sediments has lagged behind, and is still almost ex-
clusively based on knowledge derived from in vitro model systems.
Such knowledge needs to be validated with data derived from in situ
studies. In recent years, the advent of high-throughput sequencing tech-
nologies has made it possible to directly measure rates of DNA damage
that were previously calculated only by extrapolation from in vitro
experiments (Overballe-Petersen et al., 2012). A variety of bioinformat-
ics methods have been introduced to estimate DNA damage, such
as mapDamage, a software tool to map and quantify nucleotide
misincorporations due to cytosine deamination and depurination-
induced strand breaks (Ginolhac et al., 2011; Jónsson et al., 2013). To
map polymerase-blocking lesions, such as hydantoins, pyrimidine
dimers, and ICLs, a different method, called Polymerase Extension
Profiling, has been developed (Heyn et al., 2010). A well-equipped tool-
box has therefore become available to finally address patterns and rates
of DNA decay in marine sediments.
5. RNA in the extracellular environment?
Studies focusing on the active fraction of environmental microbi-
al communities often employ RNA, rather than DNA, as molecular
biomarker. The underlying assumptions are that: i) cellular concen-
trations of RNA (especially rRNA) linearly correlate with metabolic
activity, as opposed to DNA originating from all fractions of a com-
munity (i.e. the active, inactive, dormant and dead fractions); and
that ii) RNA quickly degrades when released into the extracellular
environment, due to spontaneous and RNase-mediated hydrolytic
cleavage. While the first assumption is based on physiological stud-
ies of a rather limited array of culturable strains (Kerkhof and
Kemp, 1999), and is recently being challenged in its general validity
(Blazewicz et al., 2013), the second remains untested in sediments.
The general perception of RNA as being highly prone to spontane-
ous hydrolysis (compared to DNA) is partly based on the presence of
a 2′-OH group on the ribose moiety, which, at pH N 6, can facilitate
hydrolytic cleavage of the 3′,5′-phosphodiester bond via the forma-
tion of an intermediate cyclic 2′,3′-phosphonucleoside (Oivanen
et al., 1998). However, this applies to RNA random coils, while
most types of RNA, such as ribosomal RNA, transfer RNA, viral geno-
mic RNA, and ribozymes occur in helical conformations. When ar-
ranged in single or, especially, double helices, RNA molecules are
more resistant to the 2′-OH nucleolytic attack, because of structural
constraints imposed by the helical conformation (Usher, 1972). On
the other hand, double-helical RNA is not protected from hydrolysis
by water molecules, but enzymatic assistance by ribonucleases (RN-
ases) is required in this case, as the uncatalyzed reaction is very slow.
Similar to DNA, however (see Section 3.4), RNA adsorbs onto clay
minerals (Franchi et al., 2003), and clay-bound RNA is less suscepti-
ble to digestion by RNases (Franchi and Gallori, 2005).
The sheer abundance of RNA may also enhance its persistence in
the extracellular environment. Although figures can vary depending
on growth conditions, RNA and DNA account for 20.5% and 3.1%, re-
spectively, of the dry weight of an average E. coli (Neidhardt and
193
Umbarger, 1996). In Saccharomyces cerevisiae, RNA is about 10% of
the dry weight, and DNA is only about 1% (Bresler, 2012). This espe-
cially applies to ribosomal RNA genes, which are represented in a
cell's RNA pools as many times as there are ribosomes, while only
present in a few copies in the genome. For example, E. coli and
S. cerevisiae only have about 10 and 150 copies of the rRNA gene op-
eron per genome, respectively, while the number of ribosomes is
typically three orders of magnitude higher (Bremer and Dennis,
1996; Warner, 1999). Therefore, upon cells death, most of the
nucleic acid released in the environment consists of RNA.
Indications that RNA may persist in the environment long after
the death of the source organism come from Edgcomb et al. (2011)
and Orsi et al. (2013). Their studies showed that the RNA pool ex-
tracted from deeply buried sediments comprised sequences from or-
ganisms clearly not adapted to the dark and anoxic conditions of the
deep sub-seafloor, such as plants, animals, and unicellular
phototrophic taxa. These findings suggest that RNA not enclosed in
living cells might be more stable and widespread in sediments than
often assumed.
6. Summary and outlook
The use of environmental DNA to describe the biodiversity of
present and past ecosystems has represented one of the most im-
pactful advancements in biological sciences. Molecular studies of
sedimentary DNA, in particular, have opened up a new era of explor-
ing the diversity of life at and beneath the seafloor. At the same time,
they revealed unexpectedly complex features and dynamics of this
DNA, and showed that it can occur in sediments in several different
forms (pools), some of which comprise genetic signature of formerly
living organisms.
Much remains to be learned about the quantitative and qualitative
contribution of each pool to the overall sedimentary DNA, mostly due
to methodological limitations in achieving their separate extraction. A
general consensus has arisen that a significant fraction (if not most) of
the DNA is not enclosed in living cells. Consequently, the overall extract-
ed DNA does not simply mirror the diversity of in situ, currently living
biomass, but also that of the overlying waters and surrounding lands
over different timescales. Surveys and comparisons of phylogenetic di-
versity within DNA pools have mainly been conducted on individual
samples from surface and near-surface sediments. Similar analyses
should include deep sediment strata, to reveal potential changes
down-core in the contribution of each DNA pool to the overall sediment
biodiversity. Also, studies have so far focused on established 16S and
18S rRNA genes as phylogenetic markers. However, these markers
alone can only provide a limited snapshot of the whole sequence pool,
while a direct metagenomic approach (i.e. sequencing of the bulk geno-
mic DNA without prior PCR amplification of specific marker genes)
could yield a more complete and realistic picture. Similar investigations
should also be conducted on RNA, to ascertain whether it can occur out-
side living cells and its true preservation potential in marine sediments.
The use of ancient DNA in Quaternary Sciences as biomarker for past
biodiversity would greatly benefit from a more detailed understanding
of pathways and rates of DNA decay in sediments, and how these vary
depending on the sedimentary, geochemical, and geophysical context,
the source organism, and the specific DNA pool (e.g. extracellular,
adsorbed onto minerals, or locked within dead cells or sediment aggre-
gates). Knowing what types of damages are more likely to affect ancient
DNA would allow a more accurate distinction of sequence variants
reflecting true biodiversity from those arising from damage-induced
sequencing errors. At the same time it may help design strategies to
increase success in ancient DNA recovery.
Genomic analysis of sedimentary DNA pools is merely at its incep-
tion. Methodological advancements in extracting DNA pools and appli-
cation of state-of-the-art genomic analyses are probably our best bet to
194 A. Torti et al. / Marine Genomics 24 (2015) 185–196
fully understand the origin and fate of DNA in marine sediments, and
unlock its potential for discoveries.
Conflicts of interest
The authors have no conflicts of interest to declare.
Acknowledgements
This work has been funded by the Danish National Research Founda-
tion and the European Research Council (ERC) (Advanced Grant
MICROENERGY, grant agreement no. 294200), awarded to Bo Barker
Jørgensen under the EU 7th FP.
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