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E. Capo etal. (eds.), Tracking Environmental Change Using Lake Sediments,
Developments in Paleoenvironmental Research 21,
https://doi.org/10.1007/978-3-031-43799-1_2
Chapter 2
The Sources andFates ofLake
Sedimentary DNA
CharlineGiguet-Covex, StanislavJelavić, AnthonyFoucher,
MarinaA.Morlock, SusannaA.Wood, FemkeAugustijns, IsabelleDomaizon,
LudovicGielly, andEricCapo
Keywords Sedimentary DNA · DNA taphonomy · DNA sources · DNA transfer ·
DNA degradation · DNA preservation · Lakes
Introduction
For over two decades, ancient DNA (aDNA) from various organisms has been suc-
cessfully recovered from lake sediments from all over the world, ranging from
decades to hundreds of thousands of years old (Fig.2.1). Analysis of lake
C. Giguet-Covex (*)
UMR 5204 EDYTEM, Université Savoie Mont Blanc, CNRS, Le Bourget du Lac, France
e-mail: charline.giguet-covex@univ-smb.fr
S. Jelavić
Université Grenoble Alpes, Université Savoie Mont Blanc, CNRS, IRD, Université Gustave
Eiffel, ISTerre, Grenoble, France
e-mail: stanislav.jelavic@univ-grenoble-alpes.fr
A. Foucher
Laboratoire des Sciences du Climat et de l’Environnement, UMR8212 (CEA/CNRS/UVSQ),
Université Paris-Saclay, Gif-sur-Yvette, France
e-mail: anthony.foucher@lsce.ipsl.fr
M. A. Morlock · E. Capo
Department of Ecology and Environmental Science, Umeå University, Umeå, Sweden
e-mail: marina.morlock@umu.se; eric.capo@umu.se
S. A. Wood
Cawthron Institute, Nelson, New Zealand
e-mail: susie.wood@cawthron.org.nz
10
sedimentary DNA (sedDNA) has provided new information about past aquatic and
terrestrial biodiversity and the trajectories of socio-ecosystems in various biomes
(Crump 2021). These data have enabled scientists to answer ecological/environ-
mental questions about organisms that do not leave visible and identiable remains
in sedimentary archives and that are therefore overlooked in traditional paleolim-
nology studies, e.g. sh and terrestrial mammals (e.g., Matisoo-Smith etal. 2008;
Graham etal. 2016; Domaizon etal. 2017). SedDNA analyses also provided com-
plementary information on taxonomic groups that are traditionally studied via mor-
phological analysis, e.g. diatom cysts and pollen grains (Stoof-Leichsenring etal.
2012; Parducci etal. 2015).
SedDNA based-approaches have a great potential to address key and novel ques-
tions in ecology, such as on ecosystem functioning and community structure (Keck
etal. 2020), phylogenetic and functional diversity and species richness (Huang etal.
2021; Alsos etal. 2022) and biogeographic patterns, for instance in response to
glacial retreat or human movements (i.e., colonisation, migrations; Pedersen etal.
2016; Ficetola etal. 2018). These approaches are also of high interest for archaeolo-
gists to identify past human occupations and activities (Giguet-Covex etal. 2014;
Brown etal. 2021) and their impacts on landscapes and ecosystems (Pansu etal.
2015a; Messager etal. 2022). Determining to what extent sedDNA records repre-
sent the past composition of living organisms is crucial to propose robust models
and provide reliable answers to questions of interest (Birks and Birks 2016; Alsos
etal. 2018; Capo etal. 2022). The reliability of DNA records is not only linked to
methodological aspects (see Chap. 3 of this volume) but also to taphonomic pro-
cesses leading to the record of organisms in the sediments (Giguet-Covex etal.
2019; Zinger etal. 2019; Capo etal. 2021). Taphonomy– “taphos’‘ for burial and
“nomos” for law– as applied to lake sediments include the exploration of processes
of production at the source (on land or in the water [and sediment] column), of
transportation and deposition, as well as of preservation at each step of this chain of
processes (Fig.2.2).
The understanding of the sources and fates of lake sedDNA encompasses a broad
range of research elds, from bio-geochemistry, sedimentology, biology and archae-
ology to forensic analyses, and is based on theoretical as well as empirical experi-
mental and eld studies. More specically, knowledge can be gained through the
F. Augustijns
Department of Earth and Environmental Sciences, Katholieke Universiteit Leuven,
Leuven, Belgium
e-mail: femke.augustijns@kuleuven.be
I. Domaizon
Pôle R&D ECLA, INRAE, CARRTEL, Thonon les Bains, France
e-mail: isabelle.domaizon@inrae.fr
L. Gielly
Université Grenoble Alpes, Université Savoie Mont Blanc, CNRS, LECA, Grenoble, France
e-mail: ludovic.gielly@univ-grenoble-alpes.fr
C. Giguet-Covex etal.
11
longitude (° dec)
latitude (° dec) altitude (m)
2000 2005 2010 2015 2020
2000 2005 2010 2015 2020
0
10
20
30
40
50
60
70
years
N publication
Total
Considering taphonomic issues
−40
0
40
80
−100
01
00
0
1000
2000
3000
4000
Period (years)
10000
20000
30000
40000
50000
unspecified
125kyrs
−150 −100 −50 050 100 150
Fig. 2.1 Location and altitude of lake sedimentary DNA studies and their temporality (period in
years, grey dots represent studies where the temporality is not reported in the database used, i.e.
Von Eggers etal. 2022). Trends of the number of publications, including those considering poten-
tial taphonomic issues in the DNA records, are based on the following key words (used in the
appropriate context) in the publications: taphonomy/ic, source, origin, production, transfer/trans-
port, preservation, degradation, diagenesis and reliable/bility. (Modied from Von Eggers
etal. 2022)
2 The Sources andFates ofLake Sedimentary DNA
12
Terrestrial
organisms
(incl. microbes)
Aquatic
organisms
(incl. microbes)
DNA Production
(above-ground organisms)
DNA Production
(soil organisms)
beginning of
DNA Degradation
DNA Degradation
(incl. diagenesis)
DNA Degradation &
Preservation (exDNA binding)
during paedogenesis
DNA Transfer in the catchment
via Erosion
(surface water runoff
negligible for
exDNA, but not inDNA)
Primary
DN
A sources
Secondary
DN
A sources
Soil surface
DNA Transfer in the lake
via overflows, interflows,
homopycnal currents and
settling or underflows
DNA
Degradation
DNA
Dilution
(terrestrial DNA
or Aquatic DNA
)
Transfer via
settling/in-situ
incorporation
Sediment living
microbes
DNA
Production
microbial
DNA Production DNA preservation
(exDNA binding)
DNA preservation
(exDNA binding)
Fig. 2.2 Synthesis of the chain of taphonomic processes (in teal) affecting sedimentary DNA
signals from terrestrial, water and sedimentary environments
study of DNA-mineral interactions (e.g., Kanbar etal. 2020; Freeman etal. 2023),
theoretical and experimental DNA life-time (Smith etal. 2001; Allentoft etal. 2012;
Lindahl and Nyberg 1972), erosion dynamics (Evrard etal. 2019; Foucher etal.
2020), geomicrobiology (Vuillemin etal. 2016a, 2017) and DNA releases to the
environment (Poté etal. 2009; Rourke etal. 2022). Conditions driving taphonomic
processes can vary between sites and over time, the biological group and even traits
and the type of DNA targeted (i.e., extracellular DNA (exDNA), intracellular DNA
(inDNA) or total DNA). A few sedDNA studies have addressed these consider-
ations, adding to our knowledge of their potential biases for ecological reconstruc-
tions (Boere etal. 2011a; Alsos etal. 2018; Giguet-Covex etal. 2019; Gauthier etal.
2021; Capo etal. 2017; Vuillemin etal. 2017; Kanbar etal. 2020; Jia etal. 2022a,
b) (Fig.2.1). Consequently, our knowledge of taphonomic processes and their
impacts on paleoenvironmental/ecological/climatic reconstructions is still poorly
understood. The transfer processes of DNA from the catchment area to the lake
sediments remains especially under-studied compared to degradation processes,
which benet from signicant knowledge-base acquired since the beginning of
ancient DNA studies (Pääbo 1984).
In this book chapter, we review the current knowledge on taphonomic processes
and factors driving them and affecting lake sedDNA records originating from both
terrestrial and aquatic organisms (Fig.2.2; Table2.1).
C. Giguet-Covex etal.
13
Effects ofTaphonomic Processes onDNA Fractions Archived
inLake Sediments
The total sedDNA pool is composed of intracellular DNA (inDNA) and extracellu-
lar DNA (exDNA) fractions (Torti etal. 2015; Ellegaard etal. 2020; Capo etal.
2021). InDNA is dened as being inside the cells, whether they are dead or alive.
ExDNA is released into the environment after cell lysis, also irrespective of whether
the cells come from dead organisms or organisms still alive. Although exDNA can
remain free in the environment, it usually get quickly adsorbed on minerals, organo-
mineral complexes and organic compounds such as humic substances, which sig-
nicantly increase DNA lifetime (Lorenz etal. 1981; Cai etal. 2006a; Saeki etal.
DNA origin Taphonomic processesForcing factorsSpatial variability
Inter-
species
variability
DNA production (primary
source )
Biomass and copy
number of DNA in plasts,
mitochondries…, type of biological
tissue
Yes, linked to habitats Yes
DNA degradation (incl.
during pedogenesis, and
diagenesis)
Physico-chemical conditions, UV &
biological activity, litter turnover
rates, time
YesYes
exDNA preservation via
DNA binding in soils
(secondary source)
Physico-chemical conditions,
mineralogy, organic components and
DNA conformation and size
YesYes ?
DNA transfer from the
catchment to the lake mostly
via erosion (surface water
runoff negligible for exDNA,
probably not for inDNA)
Precipitation, temperature, bedrock,
soil thickness, vegetation cover and
human activities
Yes, depend on the
connectivity between
the DNA sources and
the lake which depend
on both the
development of the
hydrographic web and
of soil erodibility
Yes
DNA transfer to the sediment
via overflow, interflow,
homopycnal current and
settling or underflow
Lake water density vs density of
sedimentary inputs and lake thermal
stratification
DNA dilution Depend on allochtonous inputs
relative to autochtonous production YesYes
DNA production
Biomass and copy
number of DNA in plasts,
mitochondries…, type of biological
tissue
Yes, linked to habitats Yes
DNA transfer Settling/in-situ incorporationYes, linked to currentsYes
DNA dilution Depend on autochtonous inputs
relative to allochtonous production YesYes
exDNA preservation via
DNA binding to particles
Physico-chemical conditions,
mineralogy, organic components and
DNA conformation and size,
availability of binding sites
YesYes ?
DNA degradation (incl.
during diagenesis)
Physico-chemical conditions, UV,
speed of sedimentation of dead
cells/carcasses in the water column
and grazing by predators
YesYes
DNA production
Biomass and copy
number of DNA in plasts,
mitochondries…, type of biological
tissue
Yes, linked to habitats Yes
exDNA preservation via
DNA binding to particles
Physico-chemical conditions,
mineralogy, organic components and
DNA conformation and size,
availability of binding sites
YesYes ?
Sediment-living
microbes
Terrestrial
organisms
Can be, especially in large
catchment and lake, with several
tributaries
Aquatic
organisms
DNA degradation (from early
diagenesis to deep burial)
Physico-chemical conditions,
biological activity & time YesYes
exDNA/inD
NA
variability
Potential issues for sedaDNA
reconstructions
Yes Over/underestimation (or even
absence) of taxa
YesLoss or poor detection of taxa
Yes
Yes
probably
partly
No Loss of the rarest taxa
Yes
Yes
No Loss of the rarest taxa
Over/underestimation (or even
absence) of taxa
Yes Loss or poor detection of taxa (may
be season dependent)
Yes Over/underestimation (or even
absence) of taxa, challenges the
understanding of microbial records
and paleoecological reconstructions
Over/underestimation (or even
absence) of taxa, integration of "r
elic
DNA", changes of DNA sources over
time not only spatially in the
catchment area but also in soil
horizons affected by erosion
Over/underestimation (or even
absence) of taxa
YesLoss or poor detection of taxa
Table 2.1 Forcing factors driving each taphonomic process affecting terrestrial, aquatic and
sediment-living organisms. Expected consequences associated with each of these processes in
terms of spatial variability, interspecies variability and exDNA vs inDNA variability and potential
issues for sedDNA reconstructions are also described
2 The Sources andFates ofLake Sedimentary DNA
14
2011). Free DNA thus should not represent a signicant fraction in the sedDNA
pool. Knowledge about taphonomic processes affecting each of these DNA frac-
tions is still limited, although some studies have shown variability between exDNA
and inDNA fractions in terms of quantity and taxonomic composition in both lacus-
trine and marine sediments (Corinaldesi etal. 2005; Torti etal. 2018; Vuillemin
etal. 2017; Case studies A3, 4, 5in Capo etal. 2021; Gauthier etal. 2022).
Most sedDNA studies use protocols to extract the total DNA pool, but some stud-
ies have focused on recovering specically the exDNA and inDNA fractions (see
syntheses in Armbrecht etal. 2019; Capo etal. 2021). Regarding the exDNA extrac-
tion protocols, only the soluble fraction is extracted by washing with alkaline phos-
phate buffers, while the insoluble fraction, corresponding to organically/
inorganically complexed DNA, remains inaccessible with this method (Torti etal.
2015). In the sedDNA literature, the most used protocol to extract exDNA is the one
developed by Taberlet etal. (2012). More recently, a modied version of this proto-
col has been developed to increase the rate of recovery (Giguet-Covex etal. 2020;
Capo etal. 2021). For total DNA extractions, more protocols are available and used
in publications but the most popular are those from the PowerMax SoilⓇ and
PowerSoilⓇ (Qiagen) kits (Capo etal. 2021). Here, we report on how both DNA
quantity and the taxonomic composition obtained from the inDNA (or total DNA)
and exDNA fractions may be differentially affected by taphonomic processes due to
different sources, transfer processes or preservation processes/conditions.
Concentrations ofinDNA andexDNA Fractions
inLake Sediments
Studies have shown that the geochemical composition of sediments, especially with
different proportions and sources of the organic matter, have an inuence on the
proportions of inDNA and exDNA fractions in the total DNA pool. For instance, in
recent (post-1950) sediments from two alpine and shallow lakes and two deep hard-
water lakes, a linear positive correlation (r2=0.98, p=0.0072) has been observed
between the total/exDNA ratio and the organic matter content (Fig.2.3a, case study
A3 from Capo etal. 2021). In this case study, sediments with less than 10% of
organic matter, showed similar concentrations of total DNA extracted with the
NucleoSpin® Soil kit (Macherey-Nagel, Düren, Germany) and of exDNA extracted
following the Taberlet etal. (2012) protocol. In contrast, the sediments with the
highest organic matter content (60%) were 7.5 times more concentrated in total
DNA than in exDNA. These results suggest that organic-rich sediments are enriched
in inDNA and are thus more suitable environments for preserving dead cells and for
supporting dormant and live cells, but not necessarily for preserving the soluble
fraction of exDNA. In that study, the researchers also observed that the most
organic-rich sediments are not the richest in DNA, regardless of the fraction tar-
geted (Fig.2.3a; Capo etal. 2021). This lack of correlation has also been evidenced
C. Giguet-Covex etal.
15
Aquatic OM Terrestrial OM
020406080 100 120
0100 200300
Depth (cm)
totDNA/exDNA
020406080 100 120
0612
totDNA (104ng/g dry sed)
DNA (ng/g wet sed)
DNA (ng/g wet sed)
020406080 100 120
510152025
C/N
020406080 100 120
0 600 1200
exDNA (ng/g dry sed)
020406080 100 120
C (%)
15 20 25 30 35 40
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
0
1
2
3
4
5
6
7
8
9
exDNA (0.75g)
totDNA (0.75g)
exDNA (4g)
exDNA (0.75g)
totDNA (0.75g)
exDNA (4g)
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
010203040506070
deep hardwater
peri-Alpine lakes shallow Alpine lakes
Organic matter (LOI 550°C, %)
totDNA/exDNA
AB
CDEF G
a
b
c
a
b
c
total DNA
exDNA
total DNA/exDNA
R2=0.98
Fig. 2.3 Relationships between the different DNA pools (total DNA (totDNA) vs. extracellular
DNA (exDNA) concentrations in ng.g−1 of wet or dry sediment (sed)) and organic matter (OM)
quantity and/or quality in temperate lakes (deep hard-water lakes Léman and Le Bourget in blue
and Alpine shallow lakes Serre de l’Homme and Lauzanier in pink; Capo etal. 2021) and in a
tropical organic-rich and shallow lake (Gelba in Ethiopia, original data). A and B present the rela-
tionship between DNA concentrations and organic matter content (loss on ignition at 550°C) from
the temperate lake sediments, and C to G represent the DNA concentrations and ratio, the organic
matter concentration (C% from elemental analyses) and the C/N atomic ratio (from elemental
analyses) from tropical lake sediments. Phases a, b and c correspond to phases discussed in the text
from tropical peat and lake sediments containing 31 to 80% of organic matter and
analysed following a similar extraction protocol as in the previous study (Fig.2.3c,
d, f; Giguet-Covex etal. 2023). However, in this study (Giguet-Covex etal. 2023),
a decreasing trend with depth of both DNA fractions can be observed suggesting a
temporal degradation over the last 2500years. As for organic-rich recent sediments
studied in Capo etal. (2021), total DNA concentrations were higher than exDNA
concentrations (21–278 times higher), suggesting that inDNA accounts for most of
the total DNA pool. However, in this case the total DNA/exDNA ratio was not cor-
related with the organic matter content. Three distinct deposition phases were
dened based on the total DNA/exDNA ratio (phases a, b, c; Fig.2.3d). Phase b,
characterised by a high ratio, mostly contains organic matter of aquatic origin (low
C/N atomic ratio; Bertrand etal. 2010; Thevenon etal. 2012), while phases a and c,
characterised by a low ratio, are enriched in terrestrial organic matter (high C/N)
2 The Sources andFates ofLake Sedimentary DNA
16
(Fig.2.3e, g). These results suggest that, in this lake, the different sources of organic
matter explains the different contributions of inDNA and exDNA to the total DNA
pool. Although the inDNA fraction is always the dominant fraction, sediments rich
in autochthonous organic material contained more inDNA and less exDNA than
sediments rich in allochthonous organic matter.
Efciencies ofExtraction Methods
Different extraction methods yield variable exDNA and inDNA concentrations and
might not recover all DNA molecules from a sediment sample (Capo etal. 2021).
For instance, in peat and lake sediments in Russia and Europe, concentrations of the
exDNA and inDNA fractions extracted by coupling the phosphate buffer protocol
from Taberlet etal. (2012) and the PowerSoil protocol were 10 times lower (com-
bined: wet sediment concentration of 58ng.g−1) than those of total DNA extracted
following the powerSoil protocol (wet sediment concentration of 557ng.g−1). This
result highlights that only a small fraction of exDNA and/or inDNA was extracted
(case study A4in Capo etal. 2021). Importantly, the extraction efciency depends
on the amount of sediment used as a template, its geochemical composition and
chemicals/kits used for extraction. In two hard-water lakes with 35–60% of carbon-
ates in sediments, the amount of exDNA retrieved increased (in wet sediment con-
centration, from ~200–1000 and 2000ng.g−1 in each lake) by increasing the amount
of sediment used for the extraction (from 0.75 to 4g of wet sediments), while no
increase was observed for the organic-rich lakes characterised by 2.3–3.5% of car-
bonates in the sediments (Fig.2.3b, case study A3in Capo etal. 2021). Carbonates,
such as calcite, are probably effective at storing exDNA because of the strong
adsorption of DNA at the edges of calcite crystals (Freeman etal. 2023). This DNA
is thus harder to extract (case study A6 from Capo etal. (2021). Dening the tapho-
nomic processes affecting the different DNA fractions remains challenging, in part
due to these methodological shortcomings. As such, future studies are required to
improve analytical and interpretative frameworks.
Molecular Inventories frominDNA andexDNA Fractions
Based on qPCR analyses, Nota and Parducci (case study A4in Capo etal. 2021)
assessed differences between the PCR amplication efciency of inDNA and
exDNA from various taxonomic groups (i.e., arthropods, bacteria, diatoms, eukary-
otes, plants and vertebrates). For bacteria, no difference between inDNA and
exDNA amplication efciency was observed. In contrast, total eukaryotes were
better amplied from the exDNA fraction, while the amplication of arthropods,
diatoms, plants and vertebrates was more efcient with the inDNA fraction. Among
these groups, only diatoms showed similar melting temperature proles (suggesting
C. Giguet-Covex etal.
17
similar diversity) in both DNA fractions. The differences observed in the melting
temperatures of PCR amplication assays between the inDNA and exDNA frac-
tions for arthropods, plants and vertebrates suggest the amplications of DNA from
different taxa. These results may reect differences in the taphonomic processes
that affect each DNA fraction from these groups, although it is difcult to assess this
presumption in the absence of sequencing analyses and information about sediment
types or mineralogical compositions. The different presumed taphonomic mecha-
nisms are summarised below.
Some aquatic organisms, such as diatoms and chrysophytes, leave siliceous
remains (i.e. diatom valves and chrysophyte scales and cysts) and dormant cells that
are preserved for long periods of time in sediments while other organisms do not.
This likely affects the taxonomic composition of DNA inventories from inDNA
compared to exDNA fractions, where protected cells contribute more to the inDNA
compared to the exDNA fraction. This might explain the better amplication (sug-
gesting higher quantity) of diatoms in the inDNA fraction than in the exDNA frac-
tion (case study A4in Capo etal. 2021). Similarly, some polymers of terrestrial
organisms, such as lignin, are better preserved than others (Boere etal. 2011a;
Yoccoz etal. 2012; Foucher etal. 2020). Woody species might thus be more repre-
sented in inDNA compared to exDNA than herbaceous species, which might
explain, at least partly, the different melting temperatures found from inDNA vs
exDNA in case study A4 from Nota and Parducci in Capo etal. (2021). It is also
possible that the transfer of inDNA is different from the transfer of exDNA from
terrestrial environments, leading to different species identied in each DNA frac-
tion. This hypothesis is based on the known interactions between exDNA and min-
erals, organic compounds, and organo-mineral particles, suggesting erosion as a
likely key transfer process of exDNA (see next sections on DNA binding and ter-
restrial transfer DNA), whereas aeolian transport, direct deposition into the lake,
and overland-ow might be more important for the inDNA transfer (Fig.2.2).
Two other studies compared archaeal and bacterial communities recovered from
inDNA and exDNA fractions in different sedimentary contexts: the ferruginous
sediments of Lake Towuti, Indonesia (Vuillemin etal. 2017) and marine sediments
from Aarhus Bay, Denmark (Torti etal. 2018). The authors compared the overlap in
Operational Taxonomic Units (OTUs) obtained from the two DNA fractions. OTUs
only found in the exDNA fraction were considered exogenous to the sediment layer
analysed, while OTUs only detected in the inDNA were considered endogenous. In
Lake Towuti, OTUs specic to the exDNA represented 40–50% of all sequences of
the exDNA pool (Vuillemin etal. 2017). They included taxa from soils, such as a
majority of Actinobacteria and some Verrucomicrobia, Solibacteres and
Alphaproteobacteria, including Pseudonocardia and Pedomicrobium (Delgado-
Baquerizo etal. 2018), but also primary (e.g., Cyanobacteria) and secondary pro-
ducers (e.g., other Alpha- and Betaproteobacteria species) in the water column. In
Aarhus Bay, a lower proportion of OTUs shared between the two fractions and a
higher number of OTUs unique to the exDNA fraction was observed in the upper
sediments (<34cm-depth) compared to deeper sediments. These results were inter-
preted as the consequence of the introduction of exogenous exDNA in the sediment
2 The Sources andFates ofLake Sedimentary DNA
18
layer of interest, which are not coming from soils or the water column as in Lake
Towuti, but coming from bioturbation in the surface sediments (Torti etal. 2018). In
signicantly bioturbated sediments, reconstructions of the paleo- community com-
position and diversity from total DNA or exDNA will be thus distorted (Torti
etal. 2018).
In addition to the importance of source type (i.e., more or less resistant cells),
origin of the organisms (terrestrial, aquatic or sedimentary for microbes) and trans-
fer mechanisms, it is crucial to consider the differential degradation effects as a
function of time. With time, it is likely that inDNA increasingly contributes to the
exDNA fraction. This hypothesis is in line with the increase in depth of microbial
DNA from taxa related to known spore- and cyst-formers, including Actinobacteria,
Clostridia and Planctomycetes, observed in Lake Towuti (Vuillemin etal. 2017).
The synthesis of studies comparing the composition of each DNA fraction for
different groups of organisms highlights the complexity of the taphonomic pro-
cesses behind the fossilisation of each fraction. Given the low number of such stud-
ies, our knowledge is still limited, and the identication of general patterns is
challenging. More systematic comparisons in different contexts and including other
sedimentological and geochemical data, are necessary to assess the complementar-
ity of the signals from these DNA pools. This knowledge can also increase the range
of information offered by sedDNA analyses and its robustness by improving the
selection of the DNA fraction to be targeted (inDNA, exDNA or both DNA frac-
tions) according to the scientic question.
DNA Degradation andPreservation intheEnvironment
After cellular death, DNA repair mechanisms do not operate, leading to the degra-
dation of inDNA by chemical reactions (oxidation, hydrolysis, alkylation and the
Maillard reaction), processes that continue outside cells, affecting the exDNA frac-
tion as well (Willerslev and Cooper 2005). The exDNA fraction is also actively
damaged by enzymes (DNases) produced by microbes or can be directly consumed
by microbes as a source of nutrients. However, exDNA can bind to particles such as
minerals, carbonaceous materials (e.g., soot and charcoal), and complex organic
compounds such as humic substances or organo-mineral complexes. The adsorption
of DNA to a surface is known to reduce its degradation (Lorenz etal. 1991;
Romanowski etal. 1991; Khanna and Stotzky 1992; Crecchio and Stotzky 1998;
Cai etal. 2006a; Pietramellara etal. 2009). The mechanisms which protect the
adsorbed DNA from degradation are not entirely clear. Protection against enzymatic
activity might be related to the modication of DNA conformation in proximity of
solid surfaces (a transition between A- and B-forms and/or modication in coiling,
i.e. quaternary DNA structure), which inhibits enzymatic recognition, and/or the
adsorption of enzymes on the mineral surfaces, effectively inactivating and prevent-
ing them from attacking the DNA (Levy-Booth etal. 2007 and references therein).
The modication of conformation upon adsorption could also protect DNA against
C. Giguet-Covex etal.
19
oxidative damage (Scappini etal. 2004). Protection is possible only if DNA is
adsorbed to particles faster than any biological, physical, or chemical degradation
process and if no desorption occurs during the lifetime of a DNA-mineral/organic
complex.
The processes of DNA degradation can theoretically take place in the DNA pro-
duction area (source), during the DNA transfer from the source to the sediment (in
the catchment and water column for terrestrial DNA and in the water column for
aquatic DNA), at the water-sediment interface and upper sediments (where micro-
bial activity is relatively high and partially led to what we call “early diagenesis”)
and during deep burial in sediments (Fig.2.2). There is no consensus about whether
DNA degradation rates are faster in the water column, at the water-sediment inter-
face, or in upper or deeper sediment layers, as this seems to depend on the environ-
mental conditions. Indeed, as presented in the following sections, physico-chemical
conditions are known to affect the rates of chemical and microbial DNA degrada-
tion and the DNA binding to particles. The mineral and organic composition and the
DNA conformation are also important factors inuencing the DNA binding.
Effects ofPhysico-Chemical Conditions onDNA Degradation
Modelling and statistical approaches have revealed a relationship between the envi-
ronmental temperature and the length of DNA fragments (Smith etal. 2001; Geggier
etal. 2011; Hofreiter etal. 2015). The rate of depurination, i.e. the breaking of the
bond between a purine [adenine or guanine] and the deoxyribose due to hydrolysis,
increases as temperature increases (Lindahl and Nyberg 1972). Based on the depu-
rination rate and a thermal history of sediment, the DNA preservation can be pre-
dicted (Smith etal. 2001). For instance, at neutral pH, physiological salt concentration
and at 15° C, DNA is expected to degrade after 100,000years, but in colder envi-
ronments this theoretical limit is extended to ~400,000years (Hofreiter etal. 2001;
Willerslev etal. 2004). High temperatures have also an indirect effect on DNA
degradation by favouring the activity of microorganisms that recycle the exDNA
buried in sediments (Dell’Anno and Danovaro 2005). The respective contribution of
the direct and indirect effects of temperature on DNA degradation is still unclear
and further work is required. In lake sediments, the lowest DNA decay rates were
associated with cold temperatures (i.e. ~4° C, the typical lake bottom water tem-
perature of deep temperate lakes) and anoxic conditions because anoxia reduces
microbial activity, thus microbial degradation (Mejbel etal. 2022). This notion of
better DNA preservation in cold conditions is so deeply embedded in the scientic
community that most sedDNA studies have been conducted in high latitude/altitude
areas or in temperate environments, with little work reported from tropical regions
(Fig.2.1). Nonetheless, recent lake sedDNA studies showed that DNA preservation
in tropical areas is possible as well. Indeed, in sediments from low-altitude lakes,
DNA as old as 5000years ago was successfully extracted (Vuillemin etal. 2016a;
Bremond etal. 2017) and perhaps as old as 1 million years (Ekram etal. 2021).
2 The Sources andFates ofLake Sedimentary DNA
20
Other sedDNA studies in African high-altitude lakes (from 1200 to 4127ma.s.l.)
also reported successful sedDNA recovery from sediments up to several millennia
old (Epp etal. 2010, 2011; Stoof-Leichsenring etal. 2012; Boessenkool etal. 2014;
Dommain etal. 2020; Tabares etal. 2020).
In addition to the chemical and biological effects of temperature and oxygen on
DNA degradation, laboratory experiments revealed that high salt concentration
(high ionic strength) and high pH limit hydrolysis, which favour DNA preservation
(Lindahl and Nyberg 1972, 1974; Strickler etal. 2015). Furthermore, environments
protected from ultraviolet radiation such as sediment pores favour DNA preserva-
tion because UV radiation damages DNA (Rastogi etal. 2010; Strickler etal. 2015).
A few eld-based studies have explored potential effects of these environmental
factors on the preservation of lake sedDNA.For instance, poor DNA preservation in
acidic environments has been proposed to explain the low recovery of terrestrial
plant DNA from sediments of two small alpine lakes (Giguet-Covex etal. 2019).
The analysis of 219 lake surface sediment samples from China and Siberia sug-
gested that neutral to slightly alkaline water (i.e. pH=~7–9) and intermediate water
conductivity (100–500 μS.cm−1) facilitated plant DNA preservation in the sedi-
ments (Jia etal. 2022a).
DNA Binding onParticles: Implications forLake sedDNA
Studies, Mechanisms andFactors
The adsorption of DNA at mineral surfaces and organic particles, and its effect on
DNA preservation, is an important aspect to consider for the analysis of lake
sedDNA.Adsorbed DNA is more difcult to degrade, suggesting that it can survive
in sediments across time and space (Sand and Jelavić 2018), likely even in warm
and humid environments depending on the mineral substrate and the nature of bind-
ing. For instance clay minerals, recognized as “DNA hotspots’‘in lake sediments
(Kanbar etal. 2020), in combination with low ambient temperatures, are likely
responsible for the persistence of ~2 million years old DNA in ancient shallow
marine near-shore sediments in Northern Greenland (Kjær etal. 2022). This study
extended by more than 1.5Ma the previous estimates of the theoretical limit of
DNA preservation based on temperature as the only determining factor. In perma-
frost and cave sediments, minerals are also suspected as important contributors to
the persistence of hundreds of thousands year old DNA (Slon etal. 2017; Zavala
etal. 2021; Wang etal. 2021). The success of lake sedDNA studies reported from
tropical areas may also relate to the presence of appropriate substrate favouring the
DNA binding and thus preservation.
For terrestrial organisms, the adsorption of DNA on particles is important to
consider because this process is expected to determine the mode of exDNA transfer
to the lake bottom. More specically, the adsorption of DNA on particles implies
that allochthonous exDNA is mostly likely to be transported to lake sediment during
C. Giguet-Covex etal.
21
erosion events and not (or to a much lesser extent) during surface water runoff
events (Fig.2.2; Giguet-Covex etal. 2019; Morlock etal. 2021). This involves
extracellular sedDNA reecting biological communities from areas where erosion
occurs. This has to be considered when interpreting past communities in terms of
paleoenvironmental/ecological changes.
Binding Mechanisms Between DNA andMinerals
The properties of both suspended minerals and dissolved DNA vary as a function of
the chemical conditions (eg. pH and ionic strength), which affect the adsorption and
binding between them. Here, we rst discuss basic principles governing the devel-
opment of surface charging on minerals and DNA, arguably the most important
parameter controlling their binding. Later, we discuss how the charging affects
adsorption and introduce other important parameters such as DNA conformation
and composition of ambiental solutions.
We focus the discussion on the charging properties of double stranded DNA
because the degradation rate of single stranded DNA is orders of magnitude higher
than that of double stranded DNA (Lindahl 1993), thereby suggesting that the
majority of single stranded exDNA quickly disappears from the archive. Theoretical
and experimental studies demonstrated that double stranded DNA adsorbs to com-
mon minerals via the phosphate backbone (Okazaki etal. 2001; Pietramellara etal.
2001; Saeki etal. 2010). The phosphate backbone of DNA being negatively charged
above a pH of ~2, the binding to positively charged minerals can happen directly
through electrostatic attraction, thereby resulting in the formation of strong bonds.
However, the isoelectric point of DNA, denoting the pH at which the whole mole-
cule is neutral in an electric eld, is somewhat higher, between a pH of 2–4.4
(Sherbet etal. 1983; Yetgin and Balkose 2015). Because the backbone surface of
double stranded DNA is dominated by phosphates, the much higher observed values
of isoelectric point compared to the pKa of phosphate are likely the result of either
protein impurities or exposure of nucleobases during the measurement. Such impu-
rities or exposure of nucleobases would be due to depurination of DNA at pH<5.
Thus, we hold that the true value of DNA’s isoelectric point is at the lower end of
reported range, closer to the pKa of phosphate.
The electrical charge of a mineral is a result of the nite sizes of crystals and
various crystal defects that reach the surface. With a few exceptions (e.g., a special
surface of clay minerals called the basal plane), the charge of mineral surfaces is a
function of the pH solution and ion concentration. Depending on the pH, mineral
charge can be either positive, negative or neutral. The pH at which the surface of
minerals is neutral is referred to as the Point of Zero Charge (PZC). At pH<PZC,
the mineral surface is positively charged and at pH>PZC it is negatively charged.
Depending on the ion composition and concentration in the solution, the surface
charge can, however, be neutralised or even reversed. For example, in karstic lakes
where the concentration of Ca2+ is high because of dissolution of calcite (CaCO3),
when the pH is neutral, the usually negatively charged quartz can adsorb high
2 The Sources andFates ofLake Sedimentary DNA
22
enough amounts of Ca2+ for its surface to effectively become neutral or even posi-
tively charged. Because the surface charge of minerals is largely controlled by envi-
ronmental factors such as water hardness and pH, the binding of DNA at mineral
surfaces will also be inuenced by the composition of environmental solutions.
Binding of DNA to a mineral can also take place on a negatively charged surface.
In this case DNA and a mineral must rst overcome the repulsive forces by adsorp-
tion of cations to the mineral surface, which then screens the negative charge and
allows the phosphate backbone to bind. Such an effect is often called “cation bridg-
ing” where a cation makes a “bridge” between a negatively charged mineral surface
and a negatively charged organic surface. Cation bridging implies that divalent cat-
ions common in freshwater such as Ca2+ and Mg2+ are more effective in charge
screening than monovalent such as Na+ or K+ since one ion can bind directly to both
the mineral surface and the phosphate backbone at the same time.
Factors Controlling theBinding onMinerals
The adsorption of DNA on minerals is a function of: (a) molecular weight and con-
formation (circular, linear) of DNA (Franchi etal. 1999); (b) composition and sur-
face properties of minerals such as defects and charge (Fig.2.4) (Jelavić etal. 2022;
Freeman etal. 2023); (c) composition and pH of the solution (Paget etal. 1992)
(Fig.2.4); and (d) presence of other organic compounds in the system (Cai etal.
2006a) (Table2.1).
The adsorption capacity of low molecular weight DNA is greater than for high
molecular weight DNA because of the lower steric hindrances between shorter
strands (Ogram etal. 1988; Franchi etal. 1999). This suggests that modern environ-
mental DNA molecules adsorb to a lesser extent to minerals than older, shorter, and
degraded DNA.The adsorption capacity of circular (coiled) DNA is higher than that
of linear DNA (Poly etal. 2000; Pietramellara etal. 2001), likely because of the
higher charge density of coiled DNA and the contribution of non-electrostatic forces
in the interaction between coiled DNA and minerals.
Clay minerals absorb an order of magnitude more DNA than other common soil
and sediment silicates (Lorenz etal. 1981; Kjær etal. 2022). One reason is that the
small (colloidal) size of clay minerals results in signicantly higher specic surface
area compared to minerals with larger particle size. Among clay minerals, some
smectites (so-called swelling or expansive clay minerals) have higher adsorption
capacity than kaolinite, illite and chlorites (non-swelling clay minerals) (Demanèche
etal. 2001; Pietramellara etal. 2001; Cai etal. 2006b; Kjær etal. 2022), likely
because they can adsorb DNA inside of their crystal structure, a process known as
intercalation (Greaves and Wilson 1969). However, only smectites with relatively
low surface charge are able to intercalate DNA (Jelavić etal. 2022). Iron and alu-
minium oxides, such as goethite and gibbsite, are less widespread and less common
than silicates (Ito and Wagai 2017) but have large adsorption capacities for DNA
(Saeki etal. 2010; Cao etal. 2011). Interestingly, these minerals are commonly
found in tropical soils (Schwertmann and Taylor 1989; Duchaufour etal. 2020),
C. Giguet-Covex etal.
23
Fig. 2.4 Schindler diagram for DNA binding: net charge of mineral surfaces and their binding
modalities with DNA as a function of pH.An electrostatic attraction (red horizontal line) between
the negatively charged phosphate backbone of DNA and positively charged minerals is possible at
low pH (pH<~4.5) for silicates (such as quartz, feldspars, and zeolites) and clay minerals (such
as montmorillonite, chlorites, kaolins, illites, smectites, and vermiculites) and at a large pH range
(pH<~10) for carbonates (such as calcites, magnesites, and dolomites) and Fe-oxides (such as
hematites, goethites, magnetites, and ferrihydrites). The screening of repulsion (blue horizontal
line), i.e. the neutralisation or charge reversal of negative mineral surface charges and negatively
charged phosphate backbone of DNA by cations (“cation bridge”), is possible at a pH above ~4.5
for silicates and clay minerals and at a pH above ~9.5 for carbonates and Fe-oxides. The median
value (vertical black line) and the range for point-of-zero charge of minerals (grey shaded areas)
are taken from Kosmulski (2011) and Oelkers etal. (2009)
where sedDNA studies are still poorly reported (Fig.2.1). Unlike clay minerals,
goethite and gibbsite are often positively charged at neutral pH, so they bind directly
to negatively charged DNA (Fig.2.4), likely resulting in much stronger binding
compared to the “cation bridge” interaction. Unlike surfaces that vary in charge as
a function of solution composition, structural defects or crystal steps represent areas
of permanent charge that also affect DNA binding (Freeman etal. 2023). Crystal
defects and crystal steps are features on crystal surfaces that usually exhibit larger
charge density compared to the surrounding, pristine surface. Freeman etal. (2023)
observed preferential binding of DNA to the crystal steps of calcite in NaCl, MgCl2
and NiCl2 solutions but no signicant interaction between DNA with the surround-
ing surface; this observation emphasises the importance the magnitude of surface
charge on DNA adsorption, in addition to its sign (positive or negative as a func-
tion of pH).
2 The Sources andFates ofLake Sedimentary DNA
24
Because the mineral charge is a function of the solution composition, it is impor-
tant to consider the role of pH and ions on the mineral-DNA interaction. DNA
adsorption capacity of clay minerals and other silicates is in general minimal at
circumneutral pH and increases as pH decreases to acidic values (Greaves and
Wilson 1969; Romanowski etal. 1991) corresponding to conditions at which DNA
is unstable in solution. On carbonates and iron oxides, DNA adsorption decreases as
pH increases (Cleaves etal. 2011; Sodnikar etal. 2021) because the mineral surface
becomes increasingly negative and repels DNA.Once the pH rises above the PZC,
the electrostatic repulsion between negatively charged minerals and DNA domi-
nates the interaction (Fig.2.4). However, DNA still adsorbs to clay minerals at cir-
cumneutral pH with the help of cations that can screen the negative surface charge
(“cation bridging”, Fig.2.4). Thus, the adsorption capacity of clay minerals and sili-
cates for DNA in the presence of divalent cations is larger than in the presence of
monovalent cations (Greaves and Wilson 1969).
The organic matter present in sediments and soils can either: (i) reduces the
amount of mineral able to adsorb DNA because both likely compete for the same
adsorption sites on mineral surfaces (Cai etal. 2006a, b; Saeki and Sakai 2009); or
(ii) does not have an effect on DNA adsorption (Saeki etal. 2008) because organic
compounds are not associated with minerals but represent a separate aggregated
phase within the soil or sediment. Such an explanation, however, is difcult to rec-
oncile with the observation that the DNA adsorption capacity of humic substances
is high (Saeki etal. 2011). These discrepancies might be explained by the extreme
molecular diversity of organic compounds in soils and sediments (Christl etal.
2000; Sutton and Sposito 2005) and the prospect that different organic compounds
will interact in different ways with minerals and DNA.
DNA Binding onOrganic Compounds andCarbonaceous Materials
Practical issues hinder a comprehensive insight into the interaction between DNA
and organic compounds commonly found in sediments, such as humic substances.
Humic and fulvic acids absorb light at similar wavelengths as DNA (200–300nm),
preventing a reliable spectrophotometric determination of DNA concentration in
solution with humic substances. Determination of DNA concentration based on
uorescence resolves this issue (e.g. Singer etal. 1997) but the specicity of various
uorophores is unknown in a range of pH values, concentrations of humic sub-
stances and ionic strengths. In addition, the solubility of humic substances abruptly
changes as a function of pH (Kipton etal. 1992), much more than the solubility of
minerals, the majority of which is considered insoluble at the timescale of a single
adsorption experiment, which usually lasts a few days.
A few carefully designed studies have shown that the adsorption capacity of
humic acids is high (Saeki etal. 2011), comparable to clay minerals that cannot
intercalate DNA.The interaction is largely electrostatic in nature (Nguyen and
Elimelech 2007), although some hydrophobic forces might also play a role (Saeki
etal. 2011). The adsorption is likely irreversible in solution buffered at a pH of 5.8
(Nguyen and Elimelech 2007), suggesting good preservation potential for DNA
C. Giguet-Covex etal.
25
over geological timescales, but it is unclear how a variation in pH would affect the
nature and reversibility of binding.
Pyrolysis of organic compounds in wildres produces carbonaceous materials
such as soot and charcoal. These materials are generally low in abundance but wide-
spread in soils and sediments (Schmidt and Noack 2000). The capacity of carbona-
ceous materials to adsorb DNA is higher at a pH ranging from 3 to 8 and when
compared to clay minerals that can intercalate DNA (Fang etal. 2021; Jelavić etal.
2022). Both electrostatic and hydrophobic forces control the binding, which is
likely irreversible (Fang etal. 2021; Jelavić etal. 2022), and thus conducive for
good DNA preservation. The signicant contribution of hydrophobic forces in DNA
binding to carbonaceous materials calls into question the suitability of common
DNA extraction techniques that are tailored for sequestration of electrostatically
bound DNA, revealing carbonaceous materials as a likely overlooked reservoir
of sedDNA.
Early Diagenesis intheSedimentary Column: Effects onDNA
Concentration andTaxonomic Diversity
Early diagenesis refers to physical, chemical and biological modications of the
sediments that occur soon after their depositions, i.e. at the sediment-water interface
and in the upper sediments. The thickness of this zone varies as a function of envi-
ronmental conditions, and is especially inuenced by oxygen concentration along
the sediment depth, which can vary from a few millimetres in productive hypoxic
systems to several centimetres in oligotrophic oxygenated systems (Kristensen
2000; Haglund etal. 2003; Vuillemin etal. 2013). Burial rates and frequency of
water mixing are other variables controlling the sediment thickness affected by the
early diagenesis.
Microbial activity directly inuences early diagenesis (Vuillemin etal. 2016b),
as such degradation rates of organic matter and DNA are signicant in sediments
affected by the early diagenesis. This is reected by a sudden decrease in the con-
centration of extracted DNA in the upper sediments (Vuillemin etal. 2017; Dommain
etal. 2020; Fig.2.3c, d). Early diagenesis is also expected to modify the DNA com-
position, i.e. the taxonomic diversity recovered by DNA analyses. The number of
sequences from some taxa may decrease and even be lost, while microbes taxa
growing in the sediments are expected to increase in the DNA record. For instance,
Vuillemin etal. (2017) analysed the changes in the relative abundance of shared
OTUs by both inDNA and exDNA fractions. They observed that in the shared
inDNA fraction there are three groups of taxa with different evolution patterns in the
upper ~30cm of sediment. The rst group containing Deltaproteobacteria,
Clostridia, Elusimicrobia, Bathyarchaeota, and Hadesarchaea increased in abun-
dance with depth. In contrast, the second group composed of Chlorobi, Anaerolineae
and Omnitrophica decreased in abundance, whereas the third group with
Dehalococcoidia, Nitrospirae, Aminicenantes, Aenigmarchaeota, and
Thermoplasmata rst increased before quickly decreasing in abundance. This
2 The Sources andFates ofLake Sedimentary DNA
26
succession of microbial populations was interpreted as reecting variations in bio-
geochemical conditions during early diagenesis (Vuillemin etal. 2016b). However,
the authors acknowledge that this interpretation assumes that the shared inDNA
only reects the growth, or decline of populations living in the sediments and does
not account for the accumulation of inactive intact cells in the sediments. Another
study tested the impact of early diagenesis on the protist DNA signal by analysing
laminated sediments from two cores sampled in a boreal lake with a time lag of
6years (Capo etal. 2017). In the deeper sediment layers (age~15–40years), the
molecular signal was very similar in the two cores (except for fungi) and was con-
sidered stable. However, for the uppermost strata (age<15years; upper 7cm),
moderate modications in the composition of the protist community were detected
probably due to the biogeochemical conditions.
Taxa-Dependent Preservation ofSedimentary DNA
The potential for DNA preservation in lake sediments depends on features that vary
between different species, some having protective resting stages (e.g., cysts, spores,
akinetes; Ellegaard and Ribeiro 2018) or resistant cell walls (e.g., diatom frustule,
lignin, pollen), thus being more resistant to degradation processes (Setlow 2007;
Boere etal. 2011a, b; Capo etal. 2015; Mejbel etal. 2022). In a 1.4 million years
(Ma) marine record, the number of chloroplast DNA sequences was found to
decrease with depth, with a strong decay within 100–200,000years of deposition
(Kirkpatrick etal. 2016). However, a few diatom phylotypes persisted for up to
1.4Ma, matching fossil diatom records. This result suggested that the frustules
favoured the preservation of diatom DNA.In addition, in fjord sediments from east-
ern Antarctica, dinoagellate DNA was found to degrade faster than diatoms (Boere
etal. 2011a). In 80–125ka Mediterranean sapropels, with mostly marine organic
matter, more terrestrial plant totDNA was recovered compared to planktonic taxa
(Boere etal. 2011b). In this case, most planktonic taxa were dinoagellates that
may leave low amounts of remains (i.e., protective resting stages), while terrestrial
plant DNA might have originated from pollen or other cells protected from degrada-
tion, which could explain the lower preservation of DNA from aquatic species rela-
tive to terrestrial plant DNA and the apparent discrepancy with the main origin of
the organic matter. However, based on the review of taphonomic processes in this
chapter, two other processes might have contributed to the preservation of the ter-
restrial plant DNA relative to planktonic DNA.The binding of plant DNA to detrital
mineral or organic particles likely promoted its preservation. These DNA-bearing
particles carried by the Nile River to the marine environment probably encountered
aquatic exDNA but could not adsorb it (and protect it) because of the lack of addi-
tional adsorption sites and/or due to unfavourable chemical conditions for the sorp-
tion. In addition, the potential contribution of the aquatic inDNA to the exDNA
fraction as a function of time and sedimentary depth was prevented because of high
autochthonous organic matter content which quickly metabolised newly released
C. Giguet-Covex etal.
27
exDNA. These alternative explanations highlight our lack of knowledge about the
taphonomic processes that can bring bias into the reconstruction of community
composition. More specically, a better understanding of the inuence of geochem-
ical and mineralogical sediment properties on the recovery of exDNA from different
groups over time is still needed (Sand etal. 2023). More systematic multi-proxy
approaches and analyses of the different DNA fractions are probably key to answer-
ing the following questions: (1) where to look (i.e., targeted lake and coring site
based on mineralogic composition and geochemical properties of the sediments);
(2) what to look for (i.e., targeted organisms, DNA fraction(s)); and (3) over which
time scale?
Terrestrial DNA inLake Sediments
Plants and mammals are the main terrestrial organisms targeted by DNA analyses
from lake sediments (Anderson-Carpenter etal. 2011; Parducci etal. 2013; Giguet-
Covex etal. 2014; Pedersen etal. 2016). However, in a study focused on mammals,
an unexpected detection of earthworms was also reported (Lammers etal. 2019).
Furthermore, soil-derived microbial communities (Vuillemin etal. 2017) and human
and animal faecal-specic bacteria (Madeja etal. 2009, 2010; Madeja 2015) have
been investigated in certain lakes. In terms of biomass in catchments, mammals are
generally less abundant than plants or invertebrates like earthworms (Lammers
etal. 2019), except in specic situations where there are high concentrations of
mammals such as on migration routes, waterholes or due to human management
(livestock farming and associated practices such as gathering in pens). This low
biomass may at least partly explain several reported failures in the detection of ani-
mals (Giguet-Covex etal. 2019; Lammers etal. 2019). In some lakes, the poor
detections of terrestrial plant DNA (Alsos etal. 2018; Giguet-Covex etal. 2019),
which were not attributed to degradation issues, highlight the need to improve our
understanding of taphonomic processes related to the sources and transport path-
ways of DNA and sediments, in order to reliably interpret DNA-based ecosystem
reconstructions. Below, we synthesise the knowledge and uncertainties on these
processes and their impacts on the sedDNA records.
DNA Production andSources
“Primary Source” ofDNA
At the level of organisms, for both plants and mammals, the sources of DNA
released to the environment remain largely unknown. For plants, potential sources
are above-ground (shoot systems like leaves, seeds) and below-ground (root sys-
tem) organs (Parducci etal. 2017). For plants, the relatively poor overlap between
2 The Sources andFates ofLake Sedimentary DNA
28
pollen and DNA records from lake sediments suggest that pollen does not signi-
cantly contribute to the sedDNA pool, and sedDNA from plants likely originated
from other plant tissues (Parducci etal. 2017; Messager etal. 2022). For mammals,
faeces, hair, skin akes, urine (Willerslev etal. 2003; Lydolph etal. 2005; Haile
etal. 2007) and decaying tissue from dead animals (Ficetola etal. 2018) are all
assumed to contribute to the DNA release in the environment, while a study from
archaeological soils in Greenland suggest that bones do not, or minimally contribute
to the sedDNA pool (Hebsgaard etal. 2009). However, other studies argue for the
opposite. For instance, inputs of fresh bones from crannogs into a lake were sug-
gested as a source contributing to the sedDNA signal (Brown etal. 2021). In Arctic
environments, where cold temperatures signicantly increase bone persistence and
where burial can take a long time, Miller and Simpson (2022) argue that bones
might be a continuous source of environmental DNA. This continuous release of
DNA may explain the time lags in estimating the extinction of mammoths from
bones compared to sedimentary DNA analyses (Miller and Simpson 2022).
Soil DNA, a“Secondary andPrimary Source” ofDNA
DNA from plant and animal tissues is incorporated into soils through pedogenesis
processes (Poté etal. 2005). This “soil DNA” represents a secondary source of lake
sedDNA.In addition, soils are a primary source of DNA for soil-derived microbial
communities (Vuillemin etal. 2017) and other soil-living organisms such as earth-
worms (Pansu etal. 2015a). Soil environmental DNA (eDNA) studies thus provide
useful knowledge about these primary and secondary sources of DNA for lake sedi-
ments. Importantly, they may provide answers to questions pertaining to: (1) the
spatial representativeness of the DNA signal at the scale of the earth surface; (2) the
vertical distribution of DNA in soil proles; (3) the potential for keeping quantita-
tive information, i.e. information linked to biomass; and (4) DNA persistence
in soils.
Spatial Representativeness oftheDNA Signals
Plants DNA metabarcoding analyses conducted on soils from high altitude/latitude
areas, temperate agricultural lowlands and tropical areas suggested that most soil
DNA is of local origin. For instance, in boreal ecosystems from Norway, which
were characterised by a mosaic of dwarf shrub-dominated heath and forb- and
grass-rich meadows, the plant community composition identied by DNA corre-
sponded with the above-ground diversity estimated from oristic surveys (Yoccoz
etal. 2012). In temperate agricultural lowland sites in France, the last crops to be
cultivated were detected in 96–100% of the studied soils and were among the most
abundant species, together with a large variety of weeds (Foucher etal. 2020). A
primarily local origin of plant DNA in soils was also identied in the Arctic ecosys-
tem in Svalbard (Edwards etal. 2018). However, in that study, as in Norway, distant
C. Giguet-Covex etal.
29
minor contributions, through the transport by wind, water or animals, were also
identied (Yoccoz etal. 2012; Edwards etal. 2018).
DNA Distribution inaSoil Prole
Few studies on microbial and mammal DNA investigated this question. In forest
soil proles, a higher diversity of microbial communities and a higher density of
prokaryotes have been found in the upper organic soil horizons compared to the
deeper organo-mineral and mineral horizons (Fig.2.5; Agnelli etal. 2004; Kandeler
etal. 2009). Differences were evident between the totDNA and exDNA fractions in
terms of microbial composition (Agnelli etal. 2004). Mammal DNA is heteroge-
neously distributed along the soil prole with generally higher content at the sur-
face. This distribution and, more precisely, the depth at which DNA is still detectable,
depends on a combination of factors: animal behaviour, physiological differences,
population density and soil texture, which can favour or limit leaching processes
and bioturbation (Haile etal. 2007; Hebsgaard etal. 2009; Andersen etal. 2012).
For example, sandy soils are more prone to leaching while clay acts as a barrier to
leaching. Areas used for a latrine showed a deeper DNA leaching compared to areas
only used, even regularly, as trails (Andersen etal. 2012).
Quantitative information from soil DNA analyses: In high latitude ecosystems,
relationships between the relative abundance or biomass of above-ground plants
and the relative abundance of the corresponding DNA sequences have been observed
(Yoccoz etal. 2012; Pansu etal. 2015b). This result shows that soil DNA analysis
??
meadow forest
forest
[Plant totDNA]
[Plant totDNA]
[Mammal totDNA]
[Prokaryote totDNA]
bacterial diversit
y
totDNA
exDNA
[Mammal totDNA]
high dejection
production and
percolation
(e.g. of urine)
low dejection
production and
percolation
Fig. 2.5 Terrestrial DNA.Plant, mammal and prokaryote DNA concentration proles through
depth and bacterial diversity in soil proles (white lines) based on the review of soil DNA literature
and knowledge on soil carbon. For plant DNA, soil surface concentrations proposed are based on
Yoccoz etal. (2012). The deeper concentrations are unknown (?), but inspired by knowledge on
soil carbon. Mammal DNA concentration proles are inspired by data from Andersen etal. (2012)
and proles for microbial DNA are made from data presented in Agnelli etal. (2004) and Kandeler
etal. (2009). The soil interface design is similar to Chorover etal. (2007)
2 The Sources andFates ofLake Sedimentary DNA
30
yields quantitative information. However, Yoccoz etal. (2012) demonstrated that
the relationships are different according to the plant functional groups. An almost
1:1 relationship was found between the biomass and DNA sequence proportions for
graminoids, whereas for woody plants, the DNA proportion was underestimated
compared to the biomass. In contrast to woody plants, the DNA proportion from
forbs was overestimated compared to the biomass. These differences might be due
to varying litter turnover rates, different root/shoot biomass ratios or different
genome sizes, copy numbers and chloroplast numbers between species. For mam-
mals, a study on soil surface layers from safari parks, zoological gardens and farms
also reported that the DNA concentrations were positively correlated with animal
biomass (Andersen etal. 2012).
DNA persistence in soils: Yoccoz etal. (2012) and Foucher etal. (2020) investi-
gated the issue of persistence of plant DNA in soils from formerly cultivated ter-
races in subalpine meadows and from agricultural soils in temperate lowland sites,
respectively. In subalpine meadows, DNA from crops (cereals and potatoes) culti-
vated ve decades earlier was still detected. In these soils, the DNA sequence fre-
quency declined with time, with a very poor detection after ~50years of crop
abandonment (Yoccoz etal. 2012). In the temperate agricultural soils, the DNA
persistence of similar crops (e.g., cereals, rapes, peas, potatoes, sugarbeets, sun-
owers, hemps, raddish) appeared to be lower, i.e. only 8years (Foucher etal.
2020). A declining trend with time was also evidenced as plants cultivated in the last
3years were found in higher proportion than plants cultivated 8years ago. However,
in currently cultivated plots and grasslands where grapevines were grown about
60years before the study, DNA from grapevines was still detected (Foucher etal.
2020). The differences in the soil DNA signal persistence found in the different case
studies can be explained by: (1) the different DNA fractions targeted by the studies
(exDNA in Foucher etal. (2020) vs. totDNA in Yoccoz (Yoccoz 2012); (2) a better
DNA preservation in colder subalpine environments; (3) the fact that soils studied
by Foucher etal. (2020) are still worked and used, and thus more frequent exposure
to oxygen might have favoured degradation processes; (4) slower decomposition
rates of high-lignin material (for grapevines) compared to cereal tissues; (5) a com-
bination of these factors; or (6) other unknown processes.
DNA persistence in soils has also been evidenced for other organisms, but the
duration of persistence and the factors inuencing it have not been fully resolved
from the studies conducted. For instance, for mammals, a study from soils in parks
and savanna enclosures identied DNA from Bactrian camels a few centimetres
below the soil surface, after their absence for 6years from the area (Andersen etal.
2012). Analysis of exDNA and totDNA from prokaryotes and fungi in a wide range
of soils with acidic to basic conditions (pH3.5 to 8) showed that “relic DNA”–
dened as exDNA and DNA from cells with compromised cytoplasmic mem-
branes– contributed up to 40% of the totDNA sequences and 55% of the total
taxonomic richness (Carini etal. 2016). Soils with low pH, electrical conductivity
and cation exchange capacity (low Ca2+, K+) showed higher contributions of this
relic DNA to the DNA pool. This nding agrees with the current understanding
about the conditions that favour binding of exDNA on minerals and its potential
C. Giguet-Covex etal.
31
long-term preservation. However, it is not known how long this relic DNA can per-
sist in soils. This question is of primary importance to assess potential biases in the
reconstructions of past living soil microbial communities from lake sedDNA analy-
ses. In other words– is there an important contribution of non-contemporaneous
microbial DNA to a sediment layer? Agnelli etal. (2007) investigated this question
from radiocarbon analyses of exDNA, but without success. Indeed, based on the
analyses of stable isotopes (13C and 15N) on both soil organic matter (SOM) and
exDNA, radiocarbon results were assumed to be affected by inefcient purication
procedures of the DNA extracts (Agnelli etal. 2007). Two purication procedures
were tested. In one case, the stable isotopic signatures of exDNA were the same as
for SOM, suggesting that the purication procedure was not efcient for the removal
of SOM. In the second case, the low Δ14C values suggested the presence of fossil
fuel-derived substances, which were used for the purication procedure and not
effectively removed. Consequently, it was not possible to estimate the age of soil
exDNA and thus estimate the DNA persistence.
What are the implications of the different knowledge acquired on soils for lake
sediment studies? The results on DNA persistence in soils, especially from plant
DNA studies, suggest that the transfer of old DNA molecules in lake sediments at
the scale of a few decades should be limited compared to the transfer of DNA mol-
ecules corresponding to the modern biomass. Consequently, the temporal sedDNA
reconstructions of land use and land cover should not be signicantly skewed. This
conclusion was also drawn from the comparison of a recent lake sedDNA record
and historical records of forestry plantation (Sjögren etal. 2017). Nevertheless,
higher persistence of some species, especially tree and shrub species (Foucher etal.
2020) and environmental conditions favourable for the preservation of cells and
DNA (such as Arctic environments, Miller and Simpson 2022), might lead to tem-
poral inconsistency in paleoenvironmental reconstructions, at least on the scale of a
few years to decades and possibly much more in specic environments as suggested
by Miller and Simpson (2022). Time lags of a few years to decades are not expected
to signicantly affect paleoenvironmental interpretations for long-term trend stud-
ies, because it also corresponds to the range of radiocarbon dating uncertainties.
The variations in DNA concentration and richness in soil proles involve that the
DNA signature found in the lake sediment ought to be different depending on the
soil horizon(s) that is/are eroded. In addition, the targeted DNA fraction (totDNA or
exDNA) may provide different signatures, especially regarding microbial commu-
nities (Agnelli etal. 2004).
Studies on plants and mammals showed that biomass-DNA quantity relationships
are existing in soils. Do similar relationships also exist in lake sediments? Alsos etal.
(2018) showed that 61% of dominant taxa, 47% of common taxa, 25% of scattered
taxa and 15% of rare taxa are detected in modern sediments from high latitude (boreal
and alpine) lakes. These results indicate that quantitative estimates of plant abundance
can be obtained using sedDNA approaches like metabarcoding. However, this study
also identied the role of the distance to the coring site on the rate of DNA detection
(see next section). Indirectly, this result argues for the importance to consider and
understand the DNA transfer processes as another driver of the sedDNA records.
2 The Sources andFates ofLake Sedimentary DNA
32
Transfer ofTerrestrial DNA andSpatial Representativeness
oftheRecords
DNA transfer mechanisms play an important role on the spatial representativeness
of the sediment DNA record. One common question when we explore community
composition from DNA analyses is “where did the taxa come from?” A possible
bias in past reconstructions is an articial over-estimation of taxonomic richness
due to an increase in the area contributing to the DNA inputs, which may be due to
the activation of DNA transfer mechanisms in new areas. As such, identifying trans-
fer mechanisms and their potential evolution is crucial to ensure a correct interpreta-
tion of the sedDNA data.
In Alsos etal. (2018)‘s study investigating 11 small (0.04 to 27ha) boreal and
alpine lakes located in northern Norway, the overall detection of taxa was 1.5 times
higher for <2m vegetation surveys than extended vegetation surveys (within 50m
from the lakeshore), suggesting that distance to the lake inuences the plant detec-
tion. In this study, a correlation between the catchment relief (i.e. the altitudinal
difference between the top of the catchment and the lake) and the total number of
plant taxa detected was also observed, which can be explained by a higher plant
diversity in catchment covering larger altitudinal gradients and/or a better efciency
in DNA transfer due to higher runoff and/or erosion processes in catchments with
more relief.
Two other studies targeting both plant and mammal DNA examined alpine lakes
with contrasting physical settings (e.g., topography, geology, hydrography, vegeta-
tion cover and land use), resulting in sediments either dominated by in-lake pro-
duction or by detrital and mostly mineral inputs (Giguet-Covex etal. 2019; Morlock
etal. 2021). The results suggest that catchments characterised by easily erodible
bedrock and steep slopes, are typically exposed to pronounced erosion dynamics
and present well-developed hydrographic web, which facilitate the transfer of ter-
restrial DNA to lake sediments. In addition, the study by Morlock etal. (2021),
which analysed continuously deposited background sediments and high-
precipitation event layers of similar age, showed that DNA-inferred species com-
position was signicantly different between the two contrasting erosion regimes
and that taxonomic richness was higher in event layers. Because it is unlikely that
species communities changed systematically across the catchment on yearly to
decadal timescales, the switch in taxonomic composition was likely caused by a
different sediment provenance between the two contrasting erosion regimes. In
other words, different parts of the catchment contributed DNA to the lake during
continuous annual sedimentation with low erosion inputs and during heavy precipi-
tation events leading to high erosion events (Fig.2.6). Erosion as a mechanism of
terrestrial DNA transfer has also been proposed in a study on swamp sediments in
Uganda (Dommain etal. 2020). In this record, based on a different analytical
approach (shotgun analyses compared to metabarcoding for the other studies),
C. Giguet-Covex etal.
33
Flood deposits type 1
Legend
Flood deposits type 2
climatic driver of erosion plant composition
from DNA analyses
pastoral driver of erosion Continuous hemipelagic
sedimentation
year t year t+1 (or<1)year t-1 (or<-1)
sediment
core
lithology
sediment
core
lithology
DNA transfer via erosion
sediment
core
lithology
soil destabilisation due to pastoral activityno pastoral activity change in pastoral practice
Fig. 2.6 Scenario illustrating that the same landscape can be represented in the sediment archive
by different taxonomic composition, as shown in the two upper successive ood deposits. This is
because different areas can contribute to erosion over time (e.g., changes in grazing areas).
Similarly, in large catchments, we can expect variability in meteorological conditions which can
affect the DNA composition. Flood deposits type 1 and 2 track oods transporting sediment from
different sources (more developed soils vs leptosols, respectively) and thus containing different
DNA compositions. The continuous hemipelagic sedimentation consists of in-lake production and
ne detrital particles that settle, forming the paleoenvironmental record
higher proportions of plant DNA and higher richness were detected in time periods
of higher detrital inputs as supported by higher sedimentation rate and input of
clastic elements (Al and Ti).
Findings from Evrard etal. (2019) and Giguet-Covex etal. (2022) from river and
oodplain sediments in mountainous areas, and the opinion paper by Frankl etal.
(2022), also suggest that environmental DNA analysis may help to identify areas of
intense soil erosion, as different source regions host specic plant communities,
which can function to create DNA ‘ngerprints’ and back-track erosion hotspots in
a landscape. Vice versa, if the geological and/or pedological contexts are contrast-
ing, mineralogy and/or organic geochemical analyses can be used to independently
trace the origin of the sediments (that is the source area(s) of erosion) and assess the
catchment representativity of the DNA signal in paleo-reconstructions (Giguet-
Covex etal. 2019). The larger the studied catchment area and the more differenti-
ated the habitats, the more biases can be expected in the reconstructed landscape
because erosion is unlikely to affect all habitats or not with the same magnitude.
However, in such a context we can expect to obtain information on the source(s)
contributing to erosion (and potentially of the drivers, e.g. Rapuc etal. 2021) and
thus, to identify the erosion hotspots through sedDNA analysis. Erosion processes
can strongly change over time according to the vegetation cover, soil evolution,
climate, and with the development of human activities (e.g. land use, and urbanisa-
tion). Organisms associated with human activities are thus expected to be
2 The Sources andFates ofLake Sedimentary DNA
34
well- recorded due to this positive retroaction (Giguet-Covex etal. 2019; Messager
etal. 2022). In such a case, a raw comparison of changes in taxonomic richness or
other diversity indexes between the pre-anthropic and anthropic phases may there-
fore be highly biased and requires careful interpretation.
However, intense erosion processes per se are not the only erosion-related factor
controlling the transfer of DNA to the lake system. The type of erosion must also be
taken into consideration (Giguet-Covex etal. 2019). Erosion processes can affect
the soil surface (such as sheet erosion) or deeper soil horizons (such as gully or
stream bank erosion). Because soil surface horizons exhibit higher amounts of DNA
(as shown for microbes and mammals and assumed for plants, Fig.2.5), sheet ero-
sion is expected to transfer more DNA to the lake sediments than erosion of deep
soil horizons (Giguet-Covex etal. 2019). This hypothesis is supported by data from
two alpine lake sediments, where more taxa were recorded when soil surface hori-
zons mostly contributed to the sediments (Pansu etal. 2015b; Bajard etal. 2017;
Giguet-Covex etal. 2019). Conversely, less taxa were found when deep soil hori-
zons contributed to the sediments (Fig.2.7).
Animal DNA is not uniformly distributed across the animal habitat, due to their
mobility in the catchment and the species behaviour (Andersen etal. 2012). Animals
kept in enclosures or folds are more likely to be detected in the sedDNA record
when compared to a more diffuse source when kept in low abundances or scattered
distributions Giguet-Covex etal. (2019). Carnivores seem more challenging to
detect through eDNA analysis due to their lifestyle and comparatively low biomass
(Lyet etal. 2021). Many terrestrial animals access water bodies actively for drinking
and swimming and may also urinate, defecate or die in the water, depositing their
DNA in more or less high quantity (Harper etal. 2019; Ushio etal. 2017). For
instance, an eDNA study on wild pigs in small waterers showed that the number of
individuals shedding into the water system can affect the DNA persistence (Williams
etal. 2018), which consequently may be reected in the sedDNA record. However,
an animal dying in the water is expected to shed a large quantity of DNA into the
water and sediments, which may not reect the actual biomass present in the catch-
ment. Yet, even though plants and animals have very different mobilities and distri-
butions, the detection probability for terrestrial mammals in river water samples
also correlated with catchment area and precipitation intensity (Lyet etal. 2021),
again highlighting the importance of runoff and erosion for eDNA detection. The
hydrological connection and/or erosion from the animal’s location or, more pre-
cisely, from locations where high animal DNA quantities are released (e.g., latrines,
trails, enclosures, etc.) is thus critical for their detection in the sedDNA record
(Graham etal. 2016; Ficetola etal. 2018; Giguet-Covex etal. 2019; Morlock etal.
2021). Importantly, animals, especially domestic herds/ocks, often promote the
erosion process and consequently can increase the probability of DNA transfer and
detection in lake sediments.
C. Giguet-Covex etal.
35
40
60
100
150
250
400
N terrestrial plant
taxa (>5reads)
HI
(mgHC/gTOC)
OI
(mgCO2/gTOC)N terrestrial plant
taxa (>5reads)
HI
(mgHC/gTOC)
OI
(mgCO2/gTOC)
4000 20003000 1000 0
higher richness
mostly soil surface
erosion
lower richness
mostly deep soil
horizons erosion
mostly soil surface
erosion
mostly deep soil
horizons erosion
higher richness
mostly soil surface
erosion
lower richness
mostly deep soil
horizons erosion
mostly soil surface
erosion
mostly deep soil
horizons erosion
0
20
40
100
400
200
700
5000
6000 4000 3000 2000 1000 0
100
250
400
Age (cal yr BP)
Age (cal yr BP)
Deep soils
Top soils
Top soils
Litter
Deep soils
Deep soils
Top soils
Top soils
Aquatic
Aquatic
Deep soils
Top soils
Deep soils
Top soils
A
B
Fig. 2.7 Holocene sediment records from two lakes ((a) Lake Anterne and (b) Lake La Thuile) in
the Northern French Alps highlight the inuence of different erosion processes, surface soil ero-
sion and deeper soil erosion, on the taxonomic richness of terrestrial plant DNA record. Erosion
sources are identied by pyrolysis Rock Eval analysis, which provides two indexes. The combina-
tion of values in Hydrogen Index (HI) and Oxygen Index (OI) allows distinguishing different
sources of organic matter as highlighted on the g. HI is the best indicator for identifying the
aquatic organic matter (highest values) and organic matter coming from top soils (medium values),
while OI is the best indicator for distinguishing the organic matter coming from deep soil horizons
(highest values). When HI is around 150 and OI around 250, erosion mostly affects the soil sur-
face, which corresponds to the highest plant DNA richness recorded (green areas). On the oppo-
site, when the erosion mostly affects the deep soil horizons (highest OI values and low HI), lower
taxonomic richness is observed (orange areas). This suggests that the number of plant taxa detected
in lake sediments may be modulated by the thickness of soil affected by erosion. (Data from Pansu
etal. (2015b); Bajard etal. (2017) and Giguet-Covex etal. (2019))
2 The Sources andFates ofLake Sedimentary DNA
36
Aquatic DNA inLake Sediments
Sources ofAquatic DNA: Production andSpatial Variability
Habitats ofAquatic Organisms
DNA from many aquatic organisms has been recovered from lake sediments, includ-
ing heterotrophic bacteria and protists, phytoplankton, periphyton, zooplankton,
zoobenthos, sh and aquatic macrophytes (cf. Domaizon etal. 2017; Barouillet
etal. 2023, for a synthesis). These aquatic organisms occupy different habitats in
lake systems and, therefore, their DNA is released in contrasted proportions depend-
ing on their source. Overall, DNA sources include pelagic (water column), near-
shore- littoral, and the benthic zone at the water-sediment interface (Fig.2.8). DNA
of aquatic biota may also enter the system from upstream tributaries (Thomsen and
Willerslev 2015; Pawlowski etal. 2018; Sagova-Mareckova etal. 2021, Fig.2.8).
Fish and plankton of pelagic ecosystems exhibit large variations in composition and
biomass depending on seasonality and the history of the system. Planktonic organ-
isms are, by denition, unable to propel themselves against a current and include
zoo- and phyto-planktonic organisms as well heterotrophic protists and prokary-
otes. Collectively, they perform many of the essential functions of aquatic systems
including oxygen production by photosynthesis, organic matter recycling, grazing,
predation, and parasitism. Littoral zones and benthic habitats also contain a wide
range of organisms with relatively high abundances of sh species especially during
spawning periods. The biomass dominance in these habitats make them hotspots for
Uppersediments
Deepsediments
Burial over time
Sedimentation
Early
diagenesis Water-sediment interface
Allochthonous
Benthic&
Pelagic
living
Micro-organisms
Macro-organisms
Viable cells
Dead organisms/
cellsDNA bound
to particles
(longer lifetime)
Free DNA
(short lifetime)
Aquatic plants
Fishes
Macroinvertebrates
Fig. 2.8 Illustration of the origin (allochthonous sources, and water and sediment living organ-
isms) and fate (after sedimentation) of microbial and aquatic DNA. (Modied from Capo etal.
2022). Both viable and dead cells contain DNA (inDNA). ExDNA may be found as free DNA
molecules and in higher amounts as DNA bound to particles
C. Giguet-Covex etal.
37
DNA production in lake systems. The littoral zone is also the habitat for aquatic
macrophytes, which are well detected in plant sedDNA records (Sjögren etal. 2017;
Alsos etal. 2018; Ibrahim etal. 2022). In addition, many protists and prokaryotic
taxa and macroinvertebrates (e.g., oligochaetes, nematodes, mussels, and craysh)
can be found in benthic environments (Vivien etal. 2019; Schenk etal. 2020).
Variability inDNA Detection Linked toBiomass andSpatio-Temporal
Distribution ofOrganisms andAbiotic Factors
The success in the detection of environmental DNA from aquatic organisms depends
on many factors related to the characteristics of the studied systems (e.g, water resi-
dence time, abiotic conditions) as well as the type of organisms studied with differ-
ent types of distribution and abundance. For instance, the recovery of sh DNA
from sedimentary archives is challenging and only a few studies to date have suc-
cessfully achieved this (Pedersen etal. 2016; Nelson-Chorney etal. 2019; Kuwae
etal. 2020; Thomson-Laing etal. 2022; Brown etal. 2022; Giguet-covex etal.
2022). This is likely related to their heterogeneous in-lake distributions and low
biomass (compared to other aquatic organisms), but may also be due to unknown
methodological issues (Huston etal. 2023;Chap. 9 of this volume). Considerable
research efforts have focused on understanding the relationship between sh bio-
mass and DNA concentrations extracted from water samples (for a synthesis, see
Barnes etal. 2014; Rourke etal. 2022) to enable historical reconstruction of sh
stocks using molecular methods. This approach has proved to be challenging
because multiple, sometimes interacting, biotic and abiotic factors preclude the suc-
cessful detection of sh DNA in sediments. For instance, the taxon examined, its
body size, distribution, reproduction, and migration patterns may all impact the
amount and location of DNA shed by the sh. Abiotic factors, like hydrological
processes, stratication and temperature, also affect the dispersal and persistence of
eDNA. Additionally, the biomass of aquatic populations varies between seasons,
with the increased temperature and organic matter release during spring having con-
sequences on their growth.
As an exercise we compare the biomass of different aquatic groups from lake
water columns. In lakes with oligotrophic to mesotrophic conditions, bacterial bio-
mass has been estimated between 17 and 530μg.L−1 while phytoplankton biomass
ranged between 2 and 4600μg.L−1 (Simon etal. 1992). Fish production data
obtained for 100 populations from 38 lakes and reservoirs ranged from 0.02 to
771kg.ha−1 (Downing and Plante 1993). In systems with limited external sources of
food for sh populations, their biomass should not, in theory, be higher than the
biomass of the plankton that serve as an important source of food for many species.
There is, however, no study that compares the sh biomass with other biological
groups in lakes, thus it is not possible to draw rm conclusions about which groups
contribute most to lake biomass. The generally patchy distributions of sh popula-
tions in lake systems result in spatial variations in their DNA concentrations in the
water column and the sediment (Itakura etal. 2019).
2 The Sources andFates ofLake Sedimentary DNA
38
In contrast, planktonic organisms are generally relatively evenly spread in the
water column and have a more homogeneous distribution. However, mixing, wind
and lake basin morphology can impact the spatial distribution of planktonic organ-
isms. Unlike planktonic organisms, where the DNA is more prone to be mixed by
water movement, the DNA of benthic organisms is expected to be localised espe-
cially for large organisms. Pawlowski etal. (2022) reported that DNA extracted
from sediment samples is inefcient at describing the diversity of macroinverte-
brates because their DNA typically represents only a small fraction of the total DNA
extracted from sediment, and only a limited subset of the macroinvertebrates diver-
sity is revealed by sedDNA metabarcoding analyses in comparison to results
obtained by conventional methods (morpho-taxonomic identication). Nevertheless,
recent improvements in molecular genetic methods offer reasonable chances in the
coming years to monitor meiobenthos from different environmental matrices
(Gielings etal. 2021).
Microbial DNA found in sedimentary archives is a mix of DNA originating from
external sources (terrestrial and tributaries), the water column and the sediment
(Fig.2.2). Tracing the (planktonic, benthic or others) origins of microbial DNA is
difcult because of the metabolic versatility of certain microorganisms and their
capacities to thrive, or at least remain viable, in a wide range of changing environ-
mental conditions. Molecular investigations of water samples revealed that certain
prokaryotic and eukaryotic microorganisms recorded in freshwater systems origi-
nated from surrounding catchment soils (Crump etal. 2012; Ruiz-González etal.
2015). A total of 58% and 43% of the bacteria and archaeal taxa from soil waters
could be detected downstream in lake waters (Crump etal. 2012). In contrast, in this
freshwater system, only 18% of the soil microbial eukaryotic taxa were recorded in
the lake waters. Similarly, prokaryotes commonly found in soils, i.e., belonging to
Actinobacteria, Verrucomicrobia, Alphaproteobacteria and Solibacteres, were
detected in lake sediments (Vuillemin etal. 2017). Certain groups of microbial
eukaryotes were more abundant in soils (i.e., cercozoans, amoebozoans and api-
complexans), while other in freshwater biomes (i.e., ciliates, chrysophytes, dino-
phytes, synurophytes, chytrids, and diatoms) (Grossmann etal. 2016). DNA from
both pelagic and benthic ciliates can also be retrieved in sedimentary archives (e.g.,
Stoeck etal. 2018; Barouillet etal. 2022).
Transfer ofAquatic DNA toSediments
The persistence and transfer of DNA molecules in the water column is impacted by
a range of abiotic and biotic factors (Rourke etal. 2022). For sh populations, their
DNA is released in the water during their life by shedding and excretion, and at their
death, with sh carcasses breaking down and sinking to the lake bottom. Turner
etal. (2015) showed that higher concentrations of sh DNA can be found in surface
sediments compared to the water column in experimental ponds and natural rivers.
However, higher detection of sh eDNA in water compared to sediment has been
C. Giguet-Covex etal.
39
previously observed (Valdez-Moreno etal. 2019). Although reasons for this dis-
crepancy remains unclear, sh life histories, biogeochemical factors, the nature and
origin of the sediment(i.e., allochthonous inputs vs autochthonous production) and
erosion events are likely important inuencing factors (Alsos etal. 2018; Giguet-
Covex etal. 2019). A commonly overlooked parameter is the composition of sedi-
ments and endogenous particles (mineral or organic) that can provide a preliminary
insight into the preservation potential of adsorbed exDNA. For example, in Lake
Bacalar (Mexico), Valdez-Moreno etal. (2019) detected higher eDNA concentra-
tions only during spring sampling when intense algal photosynthesis removes CO2
from the lake water and triggers calcite precipitation. Calcite might quickly adsorb
exDNA fragments and incorporate it in the lake sediments.
For planktonic organisms, the transfer of their DNA from the water column to
the sediments happens either when they die and sink while still within their own
carcasses (especially for larger taxa) or as exDNA. Free exDNA is small enough to
be controlled by the water current, thus not necessarily prone to sedimentation. In
contrast, given that the solution conditions favour adsorption, adsorbed exDNA will
settle with particles to the lake bottom at a faster rate, promoting its sedimentation.
Thus, differences are expected in the efciency of the DNA transfer from the water
to the sediments, depending on species morphologies and geochemical conditions
in the water column, bioturbation at the mud-water interface and the pore water
within sediments. Grazing or cell lysis of specic taxa during sedimentation into the
water column could be a cause of differential recovery in the sedDNA signal. Capo
etal. (2015) compared microbial eukaryotic inventories obtained from the water
column at 2m and 130m depth and from upper sediments of Lake Bourget (France).
This comparison showed that the poor efciency in recovering DNA from crypto-
phytes and haptophytes from sediments was attributed to the early diagenesis of
these taxa DNA within sediments. Supporting these results, comparison of DNA
inventories from the water column with sediment traps showed a higher detection of
cryptophytes in exDNA compared to inDNA and poor recovery of cryptophytes and
haptophytes in sediment trap samples (Gauthier etal. 2021). The poor detection of
these taxa in the sediments might be because they are soft-bodied algae, thus more
susceptible to cell lysis. Differential recovery of DNA from cyanobacteria was also
reported in Nwosu etal. (2021) when they compared DNA inventories from the
water column, sediment traps and sedimentary archives. Their ndings show a poor
representation of lamentous Planktothrix in sediment samples, despite their rela-
tively high abundance at 10m water depth contrasting with higher sedimentary
signal from aggregate-forming Aphanizomenon. Monchamp etal. (2016) compared
the richness of cyanobacterial taxa identied in water (microscopy) and sediments
(metabarcoding) samples corresponding to the same time period and showed that
the congruence between richness estimates varied between cyanobacterial orders.
The richness of Chroococcales and Synechococcales was well represented in the
sediments, but not well-captured by microscopy counts performed on water sam-
ples. Synechococcales and picocyanobacteria, in general, are well represented in
the sedimentary archives of deep hard-water lakes, probably due to their dominance
in this type of lakes and/or due to the calcite precipitation process that occurs during
2 The Sources andFates ofLake Sedimentary DNA
40
spring and summer and allows a rapid sedimentation and burying of organic matter
associated with the authigenic calcite ux (Domaizon etal. 2013). Overall, aside
from sediment sources and difference in sedimentation rates of allochthonous vs
autochthonous matters, as mentioned above, the speed of sedimentation of dead
cells/carcasses in the water column, grazing by predators, and DNA degradation
related to their morphological features are factors that likely explain the differential
recovery of the sedDNA signal from specic organisms.
Transfer mechanisms of DNA from aquatic macrophytes are poorly documented.
A common observation is that aquatic plants are often better detected compared to
terrestrial plants, either based on totDNA or exDNA analyses (Sjögren etal. 2017;
Alsos etal. 2018; Giguet-Covex etal. 2019; Ibrahim etal. 2022). This might be
related with the proximity, thus more direct connection, with the sediments. A study
based on a core taken from the deepest part of a relatively large peri-alpine lake at
least 500m from the shore showed different amounts of DNA sequences and PCR
replicates according to the categories of aquatic macrophytes (Giguet-covex etal.
2022). Free-oating plants were the most represented, followed by submerged,
oating leaf and lastly helophyte/immersed plants. This result was interpreted as
reecting the different dispersion capacities of each category of macrophyte because
of their rooting system as well as their mobility with currents and the size of their
habitat impacting the biomass production.
Summary
Two decades of research on sedDNA have revealed that lake sediments from all
over the world, from the tropics to the poles, preserve genetic information about
past aquatic and terrestrial organisms. DNA storage processes are different if we
consider the extracellular DNA or intracellular DNA fractions. Important biotic fac-
tors include biomass, habitats, physiological features, and mobility of the organ-
isms, as well as abiotic factors such as erosion dynamic, water currents, mineralogical
properties, geochemical conditions and the age of sediments. How these parameters
inuence DNA records in terms of their representativeness is still not fully under-
stood and the question of their relative importance remains unanswered.
Several uncertainties remain about the processes that control the fossilisation of
DNA from terrestrial and aquatic organisms. Identifying the sources, improving our
understanding of the transfer processes, and the fate of the DNA signal in sedimen-
tary archives is crucial for evaluating the reliability of sedDNA to reconstruct past
dynamics of aquatic and terrestrial organisms and ecosystems in general. Studies
specically dedicated to the understanding of all taphonomic processes will be key
over the next decades to assist in the robust analysis of sedDNA retrieved from
lakes. For this goal, more eld studies based on modern sediment core samples or
sediment traps, as well as experimental and modelling approaches, should be con-
ducted. The lake sedDNA record currently goes back to hundreds of thousands of
years, but we believe this can be pushed even further back in time with targeted
C. Giguet-Covex etal.
41
sampling based on the mineralogic properties of sediments and the geochemical
conditions of their formation. A more thorough understanding of DNA taphonomy
in sedimentary systems will help build a solid foundation for this tool to spatially
reconstruct past trajectories of terrestrial and aquatic ecosystems and their biodiver-
sity, and will help in the identication of drivers of ecosystem change from both
natural and anthropogenic stressors.
Acknowledgements We are very thankful to Marie-Eve Monchamp, Tyler Murchie, Weihan Jia,
Antony G Brown, Inger Greave Alsos and the editors for their comments on our manuscript. SAW
was supported by the New Zealand Ministry of Business, Innovation and Employment research
programme– Our lakes’ health; past, present, future (C05X1707). MAM was supported by the
Swiss National Science Foundation (SNSF, grant no. 188256). SJ was funded by the French
Government through MOPGA Postdoctoral Programme (reference number 3-5402234721) and a
postdoctoral fellowship from Labex OSUG@2020 (investissements d’avenir, ANR10-LABX56).
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Developments in Paleoenvironmental Research 21,
https://doi.org/10.1007/978-3-031-43799-1_3
Chapter 3
The Sedimentary Ancient DNA Workow
PeterD.Heintzman, KevinNota, AlexandraRouillard, YouriLammers,
TylerJ.Murchie, LindaArmbrecht, SandraGarcés-Pastor,
andBenjaminVernot
Keywords Sedimentary ancient DNA · Paleogenomics · Ancient metagenomics ·
Metabarcoding · Contamination
Introduction
Sedimentary ancient DNA (sedaDNA) is continuing to revolutionise our under-
standing of past biological and geological processes by retrieving and analysing the
ancient DNA preserved in lake, cave, open terrestrial, midden, permafrozen, and
marine environments (Crump 2021). The study of sedaDNA began in the late 1990s
(Coolen and Overmann 1998) with the rst reports of extinct animal sedaDNA in
2003 (Hofreiter etal. 2003; Willerslev etal. 2003). Since then, it has been shown
that sedaDNA can be recovered at high resolution from recent (101–102year-old)
(e.g., Capo etal. 2017) through to deep-time (105–106year-old) sediments from a
vast diversity of environments (Crump etal. 2021; Zavala etal. 2021; Armbrecht
P. D. Heintzman (*)
Department of Geological Sciences, Stockholm University, Stockholm, Sweden
Centre for Palaeogenetics, Stockholm, Sweden
e-mail: peter.d.heintzman@geo.su.se
K. Nota · B. Vernot
Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology,
Leipzig, Germany
e-mail: kevin_nota@eva.mpg.de; benjamin_vernot@eva.mpg.de
A. Rouillard
Department of Geosciences, UiT– The Arctic University of Norway, Tromsø, Norway
Section for GeoGenetics, Globe Institute, University of Copenhagen, Copenhagen, Denmark
e-mail: alexandra.rouillard@uit.no
