Abstract and Figures

DNA can be preserved in marine and freshwater sediments both in bulk sediment and in intact, viable resting stages. Here, we assess the potential for combined use of ancient, environmental, DNA and timeseries of resurrected long-term dormant organisms, to reconstruct trophic interactions and evolutionary adaptation to changing environments. These new methods, coupled with independent evidence of biotic and abiotic forcing factors, can provide a holistic view of past ecosystems beyond that offered by standard palaeoecology, help us assess implications of ecological and molecular change for contemporary ecosystem functioning and services, and improve our ability to predict adaptation to environmental stress. Ellegaard et al. discuss the potential for using ancient environmental DNA (eDNA), combined with resurrection ecology, to analyse trophic interactions and evolutionary adaptation to changing environments. Their Review suggests that these techniques will improve our ability to predict genetic and phenotypic adaptation to environmental stress.
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REVIEW ARTICLE
Dead or alive: sediment DNA archives as tools for
tracking aquatic evolution and adaptation
Marianne Ellegaard 1,11 , Martha R. J. Clokie2, Till Czypionka 3,
Dagmar Frisch 4, Anna Godhe5,13, Anke Kremp6,12, Andrey Letarov7,
Terry J. McGenity 8,Soa Ribeiro 9& N. John Anderson10
DNA can be preserved in marine and freshwater sediments both in bulk sediment and in
intact, viable resting stages. Here, we assess the potential for combined use of ancient,
environmental, DNA and timeseries of resurrected long-term dormant organisms, to
reconstruct trophic interactions and evolutionary adaptation to changing environments.
These new methods, coupled with independent evidence of biotic and abiotic forcing factors,
can provide a holistic view of past ecosystems beyond that offered by standard palaeoe-
cology, help us assess implications of ecological and molecular change for contemporary
ecosystem functioning and services, and improve our ability to predict adaptation to envir-
onmental stress.
Undisturbed lake and marine sediments are natural archives of past changes in biota and
their environment, and when dated, they offer the opportunity of reconstructing past
changes in e.g. both primary and secondary production and community composition1,2.
Analysing organismal remains in freshwater and marine sediment cores provides a long-term
perspective of ecological change and has a long history in both pure science and applied con-
texts3(Table 1). In the more traditional approaches to the palaeoecology of aquatic systems,
microfossil analysis is accompanied by a number of geochemical proxies including lipid bio-
markers, pigments and isotope composition (Table 1). The interpretation of these archives, i.e.
the science of palaeoecology, is dependent on understanding contemporary ecological controls as
well as the sedimentation environment and its context. The remains of a diverse range of
https://doi.org/10.1038/s42003-020-0899-z OPEN
1Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark. 2Department of
Genetics and Genome Biology, University of Leicester, University Road, Leicester LE1 7RH, UK. 3Ecology, Evolution and Biodiversity Conservation, KU-Leuven,
Charles Deberiotstraat 32 - box 2439, 3000 Leuven, Belgium. 4University of Birmingham, School of Biosciences, Birmingham B15 2TT, UK. 5Department of
Marine Sciences, University of Gothenburg, Box 461SE 405 30 Göteborg, Sweden. 6Finnish Environment Institute (SYKE), Marine Research Centre, P.O.
Box 14000251 Helsinki, Finland. 7Winogradsky Institute of Microbiology RAS, prospect 60-letiya Oktyabrya 7/2, 117312 Moscow, Russia. 8School of Life
Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, UK. 9Glaciology and Climate Department, Geological Survey of Denmark and Greenland
(GEUS), Øster Voldgade 10, 1350 KBH-K København, Denmark. 10 Department of Geography, Loughborough University, Loughborough, Leicestershire LE11
3TU, UK.
11
Present address: University College Copenhagen, Humletorvet 3, 1799 Copenhagen, Denmark.
12
Present address: Leibniz Institute for Baltic Sea
Research Warnemünde, Department of Biological Oceanography, Seestraße 15, 18119 Rostock, Germany.
13
Deceased: Anna Godhe. email: mell@kp.dk
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1234567890():,;
organisms, from viruses to mammals, can be preserved in lake
and marine sediments (Fig. 1). However, the degree to which they
faithfully reect changing abundance and community composi-
tion varies enormously depending on taxon preservation capa-
city4, the depositional environment, including sedimentation rate,
and distance to the depositional site (Fig. 2).
In the past decade, the elds of resurrection ecology and
environmental DNA (eDNA; Box 1) have developed to a degree
which now enables us to complement these traditional analyses
with analyses of temporal change at the genetic and genomic
levels. This has the potential to enhance our understanding of
evolutionary processes in aquatic systems and organisms. We can
now evaluate impacts of environmental stressors on both geno-
typic and phenotypic responses of individual species, as well as on
interactions between species and on whole communities. These
new approaches can thereby expand our capacity for predictive
modelling to project future change, and for impact assessments.
Resurrection ecology is the study of temporal series of revived
resting stages from dated sediment layers. Many planktic (as well
as benthic) species form dormant or resting stages (propagules),
which accumulate over time in aquatic sediments. Such sediment
propagule banks contain eggs, spores, cysts and other resilient
structures from all domains of life (Bacteria, Archaea and
Eukarya) as well as viruses. Importantly, resting stages from many
of these taxa can remain viable for centuries5,6(Fig. 3). From
terrestrial environments, a similar phenomenon is seen in seeds,
but in most terrestrial environments, time series and reliable
chronologies are difcult to achieve, as soil disturbance, due to
bioturbation for example, will obscure the age of different depths
and oxygenation will enhance organic matter mineralisation.
However, a few studies have been able to follow depth series in
specialised environmental settings such as marginal water bodies
(e.g., in soliuction lobe; ca. 150 years max. age)7and cedar
glades8. Nonetheless, continuous, temporally structured, well-
preserved archives covering multiple trophic levels and with strict
age-control are unique to aquatic sites with undisturbed sedi-
mentation. Temporal genetic signals of change can also be
analysed by extracting total sedimentary DNA (Box 1). Similar to
other biomarkers, DNA may be archived in aquatic sediments,
but as described below (see Fig. 2), the degree to which this occurs
depends heavily on deposition and preservation conditions. Thus,
sediment-archived DNA may be either extracellular, in dead
tissue/cells, or inside living organisms (within dormant propa-
gules or active microbes). Each of these sources of DNA can be
used for detecting genetic change that reects responses to
environmental change in natural populations and communities at
a range of trophic levels.
Combining approaches, and moving from single species to
interactions
So far, almost all studies based on resurrection ecology have
targeted single species (see the compilation in a special issue of
Evolutionary Applications vol. 1, 11, 2018). Here, we present the
potential for moving this eld toward the more complex level of
species interactions by documenting how we can achieve resur-
rection time-series for new groups of organisms (to ll gaps at
different levels of the food-web; Fig. 1).
Recent reviews9have covered the state-of-the-art on both
environmental and ancient DNA, and the body of work on res-
urrection ecology of single species (see summary below) was
presented in a recent special issue of Evolutionary Applications
(vol. 1, 11, 2018). Therefore, here we focus on how to develop the
real, and mainly untapped, potential benets of combining the
two approaches. We show how the two lines of evidence from
resurrection ecology and temporal series of sedimentary eDNA
complement each other, and how analysing both in the same
sediment record can give synergistic effects and facilitate much
deeper insights into past evolutionary and adaptive trajectories
and interactions.
Finally, we show how insights from more traditional palaeoe-
cology, including preservation and spatio-temporal variability in
deposition signals, can help in interpreting the genetic signals
from sediment cores. Building on these insights allows the eva-
luation of the robustness of different chronologies and the validity
Table 1 A summary of the strength and weaknesses of the different approaches to reconstructing aquatic ecosystems and
pheno- and genotypic variability over time in aquatic systems.
Method Strengths Weaknesses References
Palaeoecology based on fossil
organisms
>107year time scales No genotype information 93,94
Large datasets are available Labour intensive
Potentially quantitative Only some groups/species preserved
Geochemical (bio)markers >107year time scales No genotype information 95
Large databases available Potential for porewater mobility
High throughput Lacks the taxonomic specicity of DNA
sequences
Potentially quantitative
Sedimentary eDNA timeseries So far, ~105year time scales No direct phenotype information See references
in textCover all domains of life So far, above population level
High throughput Few reference sequences
Sequence data has the potential to link
specically to taxa or traits
Potential for porewater mobility
Potentially quantitative (qPCR; so far
only ~100 years)
New bacterial and archaeal signals overprint the
paleo-sequences, due to in situ growth
Risk of chimeras & contamination
Risk of bias in extraction
Resurrection ecology So far, 101-102time scales (much longer
for Bacteria and Archaea)
Labour intensive See references
in text
Linking genotype and phenotype directly Only some species preserved
Applicable at population level Potential bias in survivability, but single-cell
approaches possible
Potentially quantitative
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of the emerging signalsto what extent do they represent real
change and to what extent are they affected by preservation and
taphonomic processes?
eDNA analyses can greatly expand the taxonomic coverage of
traditional palaeoecological reconstructions, and of resurrection
series. It can also be used to specically target signals from res-
urrection model-species and to expand the range of coverage
temporally. Resurrection ecology, on the other hand, adds the
dimension of phenotypic and physiological response to the
genetic information, and enables tracking of adaptive trait shifts.
The two types of data thus offer information on different frac-
tions of past communities.
We argue that judicious application of these emerging methods
in resurrection ecology and sedimentary eDNA make it possible
to track impacts of environmental change on aquatic ecosystems
at centennial, and even millennial, time scales, and to link tem-
poral phenotypic with genotypic responses, thus enabling us to
assess adaptibility, resilience and evolvability across whole eco-
systems. We highlight the opportunities that are resulting from
the rapid development of sophisticated methodology but also
discuss the current limitations that need to be addressed in order
to achieve the full potential of temporal reconstruction of genetic
and phenotypic responses to environmental change.
Resurrection ecology: studies on temporal series of intact
cells/propagules
Dormant stages of many species can remain alive in aquatic
sediments for decades and even centuries10,11,12 (see examples in
Fig. 3), and it is possible to resurrectpast populations from
these resting stages buried in undisturbed sediment via hatching
of zooplankton eggs or germination of phytoplankton cysts and
spores (Box 1), and potentially spores and other dormant forms
of Bacteria, Archaea, Fungi, as well as, potentially, other uni-
cellular heterotrophic organisms. Resurrection ecology, which is
the science based on testing temporal, revived, series of strains,
now encompasses several trophic levels of the aquatic food web
(Figs. 1and 3) and can vastly increase our understanding of
responses to changes at both phenotypic and genotypic levels. In
addition, DNA can be obtained from resurrected strains in a
quantity and quality that make in-depth analysis of full genome
sequences feasible, providing enormous potential for insights into
evolutionary genomics13. Genomic information from living pro-
pagules will be a key resource for reconstructing marine and
freshwater biological community responses to environmental
change, e.g. by discovery of loci of adaptation by applying
genome-wide association studies. Resurrection studies further
allow reconstruction of past phenotypes in the laboratory to
directly study historical populations, thereby assessing trait
changes over time as well as their transcriptomic basis14. Resur-
rection ecological studies thus occupy a unique niche by doc-
umenting actual processes of century-scale adaptation at both
genetic (Fig. 3) and phenotypic levels. So far, almost all studies
based on resurrection ecology have targeted single species and, to
date, there has been a preference for studying large, identiable,
long-lived resting stages such as Daphnia eggs and dinoagellate
cysts. Below, we document the potential for achieving resurrec-
tion time-series for new groups of organisms, to ll gaps in food-
chain levels, and discuss the potential for expanding resurrection
ecology to include interspecic and trophic-level interactions. We
thus argue that reviving resting stages from multiple organism
groups and trophic levels from the same site can now make it
Fig. 1 A schematic food-web in a limnic and coastal marine system. This gure shows where we have data on resurrection ecology series and indicates
the food-web and interaction gaps in in the palaeoecological record, which sed-eDNA has the potential to ll in, to reconstruct food-webs, possibly through
association networks, as suggested by16 for lake ecosystems. Extracting, respectively, DNA and live propagules from dated sediment core has the potential
to greatly enhance both the amount and types of information that we can gain about evolutionary and adaptive processes, by lling different information
gaps in the paleo-ecological record. Green stars indicate the organism types for which genetic and phenotypic time-series have already been established
from resurrected resting stages; the more stars, the larger the existing dataset. A green star in parenthesis indicates that viable resting stages have been
recorded from old sediment layers, but no timeseries data published. Red circles indicate organism types for which there is information based on
morphological remains (traditionalpalaeoecology); the more circles, the larger the existing dataset.
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possible to reconstruct interactions and co-adaptation trajectories
over evolutionarily relevant time scales (Fig. 1).
Virusespotential for resurrection series. Viruses are a ubi-
quitous component of the biosphere associated with other life
forms in all ecosystems. Lakes and their sediment have abundant
viral communities which are are dominated by bacteriophages15
and the sediment documents the past microbial populations from
the over-lying water body. Importantly, valuable information on
virus biology contained within this sediment archive can be
revealed from the direct resurrection of the viruses, for example
by isolating phages that infected the cyanobacterium Microcystis
up to 50 years ago. Recently, Baltic Sea sediments were found to
harbour diverse, allochthonous and autochthonously produced
viruses down to the deepest point investigated of 37 m, repre-
senting about 6000 years since deposition16. The extensive lit-
erature documenting phage particlesmorphology from deep-
sediment layers suggests that the phages are intact16, and so if the
correct host strains are available, these viruses could be cultured.
Past populations of viruses and their hosts could thus be resur-
rected and used to infer co-evolutionary dynamics over time.
Bacteria, Archaea and heterotrophic protists. Longevity of some
microbes over millions of years is now widely recognised1719.
Resurrection of Bacteria and Archaea is thus not new but differs
from studies on larger, morphologically conspicuous organisms in
that, usually, enrichment culture targetting a range of taxa is used
rather than rst separating individual organisms. Even though
bacterial palaeoecological resurrectionstudiesstartedin1990,using
(allochthonous) bacterial spores as palaeo-indicators of agricultural
land use20, and resting stages (akinetes) of cyanobacteria have been
germinated from old sediment layers21,22, the full potential of this
strategy has yet to be realised. Wunderlin et al.23 conrmed the
value of spore-formers (using non-resurrection approaches) in that
their abundance relative to total bacteria increases with sediment
age. Still, our understanding of the potential for DNA preservation
in, as well as survival of, resting stages of different bacterial taxa, is
inadequate6, and the plethora of techniques to improve microbial
cultivation and selectively enrich for specic functional groups has
barely been used in palaeoecology. Heterotrophic protists, such as
agellates, cilates and amoebae, may also be present in sediments,
both in encysted and actively metabolising states. A few studies have
cultivated amoebae found downcore in aquatic sediments24,and
other studies have shown that ciliates may survive tens of thousands
of years in permafrost soils25. However, to the best of our knowl-
edge, no genetic and/or phenotypic studies of these organisms have
been carried out on sediment time-series.
Phytoplankton. Resting stages are found in many species and in
most groups of phytoplankton12, but so far resurrection ecolo-
gical studies on time series have been restricted to a few, marine,
species: the dinoagellates Alexandrium spp., Pentapharsodinium
dalei (example in Fig. 3), and Apocalathium malmogiense26 and
the diatom Skeletonema marinoi. These have been used to explore
the impact of environmental conditions (e.g. salinity6,27,28 and
eutrophication29) on population genetic dynamics over multi-
decadal time scales, as well as phenotypic adaptation to changed
environmental conditions (e.g.6,26,27). These examples illustrate
that it is possible to trace evolutionary change of genotypes as
Fig. 2 Schematic illustration of change in relative abundance of DNA due to taphonomic processes. This gure illustrates the processes affecting DNA
distribution, degradation and/or preferential preservation during the transitions from the pelagic to the benthic zones, and from the surface sediment to the
deeper sediment. The approximate timescales of preservation of different fractions of the sediment record is also illustrated.
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well as phenotypic reaction norms of different traits in response
to environmental state using monoclonal cultures established
from revived resting stages and kept in the laboratory side-by-side
with modern strains. These phenotypic responses can then be
linked to the corresponding genomic data.
Metazoa, mainly zooplankton. The most comprehensive dataset
of resurrection ecology is probably that from crustaceans with
dormant stages. The bulk of these studies have been carried out
on the water ea Daphnia, e.g. to identify species invasions30,31
and to track effects of eutrophication32,33 (example in Fig. 3).
Many other invertebrates are present in propagule banks but have
rarely been exploited for genetic or resurrection studies34. How-
ever, Epp et al.35 were able to relate changes in different geno-
types of Brachionus rotifers to dramatic environmental change
such as water level or the deposition of volcanic ashes over 100
years. Using dormant copepod eggs retrieved from lake sediment,
Makino et al.36 recovered 21 haplotypes using 28S ribosomal
DNA. The brine shrimp Artemia is also being explored as a
model for resurrection ecology in higher salinity environments37.
Resurrection ecological studies on Daphnia spp. have shown how
a population evolved (and subsequently lost) its resistance to toxic
cyanobacterial blooms over a few decades38, documented a historical
change in the phenotypic plasticity of phosphorus physiology in
response to anthropogenic eutrophication5and showed changes in
phototactic behaviour39 and other traits40 in response to changes in
predation pressure. The comparison of transcriptomic responses of
resurrected 10- and 700-year old Daphnia isolates allowed
identication of gene networks and key functional drivers involved
in the evolutionary adaptation to eutrophication14. Recently, the rst
attempts to perform whole-genome amplication of DNA from
dormant stages have been made41a method that potentially
facilitates whole-genome sequencing even of propagules that cannot
be hatched or germinated.
For sh, aDNA has been extracted from remains of otoliths42
or scales43 from museum archives, but, although these remains
can accumulate in the sediment, we are not aware of the use of
sediment-buried sh remains to reconstruct historic evolutionary
adaptation to an environmental change at the genetic or
genomic level.
Next steps. Potential pitfalls in the study of resurrected time series
regarding e.g. issues of the non-representative nature of revived
populations4446 and differential survival can be addressed in the
planning phase of a study. Thus, biases and artefacts related to
specic phenotypes can be detected by the analysis of multiple
sediment cores39,47, cores from greater depths or from anoxic
sediments not exposed to early cues for germination/hatching. In
the case of phyto- and zooplankton resting stages, rapidly devel-
oping single-cell sequencing approaches may help to identify
germination and survival biases48, and the application of benecial
bacteria and their (or other) extracts may enhance germination49.
Issues of adaptation to culture conditions50,51 can be cir-
cumvented by phenotyping the cultures soon after germination.
Currently the main datasets for zooplankton originate from
lakes while the main datasets for phytoplankton derive from
marine coastal embayments. However, as mentioned above and
illustrated in Fig. 1, this bias can be overcome by intensifying the
effort to germinate multiple species from different trophic levels
from the same site, allowing the evolutionary dynamics of biotic
interactions to be studied if both organisms deposit viable resting
stages in the same sediment. One of the few studies to adopt this
approach revealed co-evolutionary dynamics between D. magna
and its microparasites by resurrecting host and parasite
populations from different time periods47. Similarly, the inuence
Box 1:
|
Terminology
Resurrection ecology is a rapidly evolving eld of research and consists of reviving long-term resting stages from sediment archives and using these to
create time-series of culture strains, that can be used to quantify both genetic and phenotypic response (a recent special issue in the journal Evolutionary
Applications (vol. 1, 11, 2018) is dedicated to this eld).
Resting (or dormant) stages are produced during the life cycle of a range of plankton species, as well as sediment- and soil-dwelling Bacteria, Archaea
and and heterotrophic protists, and include spores, cysts, eggs and other poorly described structures found in Bacteria and Archaea. They are
characterised by resistant walls, and a reduced metabolism, arresting further development. A mandatory state of resting is termed dormancy, or, in
zooplankton, diapause, whereas a reversible state (dependent on external stimuli) is termed quiescence (see e.g. Radzikowski36).
Resurrection is the experimentally induced reversion of buried resting stages from their inactive state of reduced metabolism to an active state of
growth and reproduction. In Bacteria, Archaea, Fungi and phytoplankton the process is termed germination, where active cells emerge or directly grow
from resting stages (e.g. akinetes of cyanobacteria, spores of bacteria and diatoms or cysts of dinoagellates and chrysophytes). Zooplankton can
hatch from dormant eggs, resuming growth and reproduction. Germination and hatching of historic resting stages are typically triggered by a change
in environmental conditions including oxygen, light and temperature (see e.g. Ellegaard and Ribeiro12).
Aquatic propagule banks are natural reservoirs of viable plankton resting stages deposited in bottom sediments. Such banks have a large signicance
for community and population dynamics as well as for the evolutionary resilience of species to rapid environmental uctuations.
DNA in aquatic sediments may originate from both dead and living organisms, and may be either allochthonous (transported) or autochthonous
(produced in situ). In this paper, we focus only on DNA produced by aquatic organisms (autochthonous). DNA in aquatic sediments may fall within
several loosely dened terms, including environmental (eDNA), sedimentary (sedDNA), ancient (aDNA) and bulk DNA, and may include either
extracellular and intracellular DNA, or a combination of both. In most cases, eDNA extracted from bulk sediment will be a combination of genetic
material in metabolically active cells and released from dead cells, while others refer to eDNA as the genetic material obtained directly from the
environment (including sediments), without any obvious sign of biological source material96. These denitions do not specically account for DNA
preserved in sediments inside structurally intact cells with varying degrees of metabolic activity, such as those of phytoplankton and zooplankton
dormant stages. Structurally intact cysts, spores and eggs in aquatic sediments may be either dead or physiologically active, and still preserve valuable
DNA material. If DNA is extracted from bulk sediments, it will have a mixed origin from both inside (possibly living and dead cells) and outside cells.
DNA obtained from resting stages (either directly or after resurrection) has been referred to as aDNA by some authors. aDNA is a term typically
applied to DNA recovered from ancient remains of dead organisms (often bones, hair, etc. but also ancient environmental samples). Others dene
ancient DNA on the basis of the quality of the DNA rather than the age97, meaning that historical DNA with no signs of fragmentation or degradation
is not aDNA by this denition. The application of this term is therefore controversial when referring to DNA obtained directly from viable resting
stages, or from resurrected strains, but may be correct when referring to DNA recovered from non-viable resting stages. As the demarcation between
dormancy and death is not easily assessed in resting stages, this terminology deserves careful consideration. Here, we use the term sedimentary-
environmental DNA (sed-eDNA) for bulk DNA extracted from dated sediment layers.
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Fig. 3 Resurrection ecology can be used to generate time series of population genetic data to test hypotheses of adaptation to temporal stressors. The
same strains, from which the DNA was extracted, can be used for side-by-side, or common-garden, tests of phenotypic/physiological response to the
same stressors. Here we show modied versions of gures from Lundholm et al.29 (a) and Frisch et al.5(b), both showing population structure plots.
aAnalysis of population genetic response of the phytoplankton Pentapharsodinium dalei in a Swedish fjord to environmental change associated with
changes in the index of the North Atlantic Oscillation (NAO), which affects, among other things, salinity and water-column stability. bAnalysis of
population genetic response of the herbivore Daphnia pulicaria in a lake to changing phosphorus concentrations through time.
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of viral infections on the changing abundance of cyanobacteria
such as Microcystis in eutrophic lakes could be shown by linking
pigment concentration with estimates of cyanophage infection
(sensu Hargreaves). The multi-faceted aspect of eutrophication
on freshwater foodwebs and species interactions has been
addressed by coupling resurrected Daphnia with bacterial
infections (Pasteuria ramosa) against a background of changing
food quality associated with increased nutrient load. Reyserhove
et al.52 show that genetic differentiation in Daphnia is affected by
food availability, and ultimately inuences parasite virulence.
Finally, an example from the marine realm: as described earlier,
resting stages of the dinoagellate Pentapharsodinium dalei can
survive ca. 100 years in undisturbed sediment cores in temparate
and arctic regions27.Parvilucifera is a genus of well-known
parasites that are strain-specic for P. dalei53. Germinating
infected strains of the dinoagellate from separated time-slices,
isolating the parasite, and attempting to re-infect strains with
parasites of a different age can give novel insights into co-
evolution in real systems at multidecadal to century time scales.
Going further, adopting a comparative genomics approach will
improve our understanding of adaptation to environmental
change by targeting specic genes or genomic regions across
taxa present in the sediment archive and linking this to specic
phenotypic responses.
DNA sedimentary archives and their taphonomy
The potential of using eDNA sediment time series in ecology was
recently covered in a thorough review by Balint et al.9. Therefore,
here we focus specically on two points: (1) the potential for
linking eDNA data with resurrection ecology, and (2) metholo-
gical issues that need more attention to tap the full potential for
reconstructing planktic communities and interactions.
The taphonomy of DNA. As mentioned in the Introduction,
valid interpretations of environmental signals derived from
aquatic sediment timeseries rely heavily on an understanding of
sedimentological processes (e.g. depositional environment and
rates) and process of preservation and degradation (taphonomy).
These considerations, perhaps even more critically, apply to the
analysis of DNA archived in aquatic sediments. Thus, a thorough
understanding of the factors inuencing DNA preservation must
underpin the eld of research into sediment timeseries of eDNA.
The probability of enzymatic and abiotic degradation of DNA
increases with time, hence the importance of rapid burial. This
particularly applies to extracellular DNA, but even intracellular
DNA will be damaged if the cell has ceased active repair. In the
dark and anoxic conditions typical of benthic sediments,
hydrolysis (depurination and deamination) is likely to be the
main abiotic process contributing to DNA decay54. Anoxia55,
minimal bioturbation55 and low temperatures56 are conducive to
an excellent sedimentary DNA archive. Extreme salinity also has
an impact on DNA preservation, as shown for brines from deep-
sea anoxic hypersaline lakes57, especially those rich in chaotropic
salts which can be effectively sterile and therefore excellent for
preserving biomolecules58. Furthermore, hardwater lakes provide
good preservation due to calcite formation, which supports rapid
sedimentation59.
A large fraction of sediment DNA is extracellular, e.g. 90% in
deep-sea sediments60 and 31% in lake sediments61. In fact,
extracellular DNA in aquatic sediments is the largest global
reservoir of DNA, with implications for ecosystem functioning60
as an important source of carbon, nitrogen and especially
phosphorus. Many microbes assimilate these elements from
DNA62 and in some cases depolymerised DNA is used as a source
of energy62,63.
Extracellular DNA has differential bioavailability, depending
on whether it is free or adsorbed to sediment particles. DNA
bound to minerals or organic matter can constitute the majority
of extracellular DNA (e.g. >95% in marine sediments64), and
attachment can enhance its preservation. Cation bridging of DNA
to clay minerals is a major mechanism by which DNA
bioavailability is reduced, due to the large surface area to volume
ratio of clay minerals and, for some clay minerals, their laminar
structure, whereby DNA can be adsorbed between clay layers54,65.
In addition to reducing bioavailability of DNA, nucleases adsorb
to minerals66, potentially reducing their activity54. Extreme
conditions may facilitate preservation of cells/DNA over
hundreds, thousands or even millions of years, as found with
evaporite minerals, such as halite17. There is still a lot to learn
about how mineral type and organic matter composition, coupled
with other factors such as ionic composition, inuence the early
diagenetic processes of extracellular sedimentary DNA54,65.
Temporal extent of DNA archives in aquatic sediments. These
preservation issues non-withstanding, DNA signals of a large
variety of organism groups have been traced continuously over
millenial time scales in aquatic sediment archives. Most studies
have extracted bulk DNA from the sediment without distin-
guishing between DNA inside live cells/resting stages and extra-
cellular DNA. Our focus is on the potential for reconstructing
planktic aquatic ecosystems and we refer to other studies for
catchment-derived DNA signals (of e.g. trees and other vegeta-
tion) stored in lake sediments67.
With regard to eukaryotic aquatic organisms, temporal changes
in sed-eDNA have been documented over time scales of
thousands to tens of thousands of years. For lakes this was
reviewed by Domaizon et al.59. In a marine setting, Lejerzerowicz
and co-workers68 recovered DNA from deep-sea sediment cores
collected in the South Atlantic, dated to about 32.500 years ago,
including DNA from taxa that do not fossilise well and
undetermined taxa. A more recent study used sed-eDNA to
estimate the colonisation date for white sh in a Swedish lake69.
eDNA approaches therefore offer the potential to assess the
impact of environmental change across taxonomic groups, over
long temporal scales, and potentially with a taxonomic resolution
unavailable by traditional microfossil approaches. However, many
authors have also highlighted limitations70 (see also Fig. 2) and
questioned the reliability of DNA archives from sediments as a
stand-alone proxy71. Instead, most researchers advocate a
combination of DNA evidence and palaeoecological approaches
as the way forward (i.e. using DNA as one proxy within a
multiproxy study)70,72,73. Indeed, as indicated in the previous
section, the mechanisms of DNA preservation are sometimes
unclear and even counterintuitive. This point is illustrated by a
sediment core study74 from the stratied Watts Basin in
Antarctica, which reported a 10-fold decline in diatom DNA
and a 10,000-fold decline in dinoagellate DNA over 2700 years.
However, there was no ecological explanation for this difference,
and quantication of the dinoagellate biomarker, dinosterol, did
not support this massive decline. Therefore, these ndings were
attributed to preferential preservation of diatom DNA within
resting stages, which were also found in the sediment record,
together with potentially greater lability of dinoagellate DNA
due to their lack of histones74. Applying parallel studies of the
two types of temporal genetic signals derived from eDNA and
resurrection ecology can further illuminate these issues.
Bacteria, Archaea, Fungi and Viruses. Bacteria, Archaea and to a
lesser extent Fungi, pose both an opportunity and a threat to the
eld of sed-eDNA. The opportunity is that, as for other organisms,
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COMMUNICATIONS BIOLOGY | (2020) 3:169 | https://doi.org/10.1038/s42003-020-0899-z | www.nature.com/commsbio 7
their sed-eDNA can be used for temporal reconstruction of
communities, with at least two requirements: rstly, that the
environment is favourable to preservation (Fig. 2); secondly, that
the DNA is from a group that would be present and functional in
the water column but not the sediment. The threat comes from the
ability of many microorganisms to function in sediments, with
two primary, interconnected effects: (1) they increase the bioa-
vailability and degradation of organic matter, including DNA and
other biomarkers derived from supposedly archived organisms;
(2) they multiply in the sediment, and thus alter the microbial
community composition, i.e. mixing up the modern (auto-
chthonous) and ancient sed-eDNA. With increasing depth and
time, most DNA derives from autochthonous microbes that are
adapted to the present deep-sediment conditions in both marine75
and lacustrine76 environments. However, exceptional preservation
may occur as discussed earlier, for example in the case of 217,000-
year-old DNA derived from phototrophic Chromatiaceae77 and
2700-year-old DNA from green-sulfur bacteria (Chlorobium)74.
Naturally, most bacterial sed-eDNA studies have focussed on
phototrophs that are allochthonous to the deep sediment,
including cyanobacteria78,79,Chlorobi74 and Chromatiaceae80.
Phylogenetic genes are valuable when there is a near-
unambiguous taxonomy-trait relationship, e.g. as found in
cyanobacteria, where photosynthesis is a group trait, notwith-
standing the capacity for other modes of energy generation in
some cyanobacterial lineages such as those detected in the deep
subsurface81 and per-alpine lakes82. More recently, genes
encoding enzymes with a specic function are being used, such
as a cyanobacterial gene (mycA) coding for the synthesis of the
toxin microcystin, which has been used to identify where
potentially toxic cyanobacterial blooms had occurred in perial-
pine lakes78. Similarly, the particulate methane monooxygenase
gene (pmoA) in anoxic lake sediments has been used to infer past
aerobic methane oxidation in the water column83. DNA extracted
from Bacillus spores in a lake sediment archive has shown a rise
in abundance of antibiotic resistance genes from the 1960s for
tetracycline resistance and 1970s for sulfonamide resistance,
demonstrating the value of such studies in understanding the
historical legacy of antibiotic use84.
Analysis of sed-eDNA has the potential to provide a unique
window into host-parasite evolution, such as that between
cyanobacterial Planktothrix chemotype hosts and their parasitic
chytrid fungi85. The lake sediment record showed stable co-
existence of host and parasite, raising questions about spatial
avoidance (infection refuges) or evolution of resistance chemo-
types in Planktothrix85. The diversity and population dynamics of
sediment viruses could also be assessed using metagenomic,
culture-independent methods by probing specic groups. Many
bacterial taxa are associated with particular phage groups, so their
diversity can be explored using primers specic to conserved genes
such as the capsid protein or portal protein genes that are currently
used as a tool to assess marine cyanophage diversity86. The phage-
encoded genes for bacterial metabolism proteins may also provide
clues to deciphering past ecological conditions. For example, some
cyanobacterial phages harbour the genes for phosphorus uptake
proteins87. A third way to explore past viral dynamics is possibly
the newly developed method that enables virus sequences to be
linked to bacteria using the frequency and abundance of CRISPR
sequences in both the viruses and bacteria88.
Next steps. Increasing phylogenetic coverage (including reference
sequences) and amount of sequence data and implementing links
to phenotypic change by targeting functional genes will help us
towards the goal of temporal reconstruction of entire ecosystems
and their response to change,
Applying multiproxy approches, and building on insights from
palaeoecological studies can give an increased understanding of
preservational issues and sampling techniques, leading to more
robust and reliable interpretations of past changes.
The benets of synergy: combining sed-eDNA and
resurrection ecology
It is apparent from the summaries above of recent work, and
other reviews, that these two new elds complement each other,
and can greatly enhance traditional palaeoecological information
derived from traditional approaches. However, to reach their full
potential, knowledge gaps and methodological concerns must be
addressed. In this nal section we briey highlight the exciting
potential of merging and expanding the analyses of genetic
archives to ll the missing ecological links indicated in Fig. 1.By
integrating investigations on multiple trophic levels, and com-
bining them with sed-eDNA analyses, the eld of resurrection
ecology can be taken a step further toward reconstructing whole-
ecosystem responses to environmental change. So far, most
experiments on eco-evolutionary dynamics spanning different
trophic levels have used simplied systems such as prey-predator
dynamics in well-controlled experiments89. The methods pre-
sented here have the potential to extend this approach to the
greater complexity of natural systems. In addition to the examples
provided in this paper, here we briey present examples of such
interactions, where temporal trajectories and species interactions,
based on existing knowledge, could be reconstructed from sedi-
ment sequences.
Most eDNA studies lack even an indirect link to phenotype
and therefore cannot connect genetic to phenotypic changes,
although functional gene analysis provides this opportunity in
some cases. In addition, there may be uncertainty associated with
the provenance of the extracted sequences. This problem is
especially true for non-model organisms for which many func-
tional pathways are unknown, or gene annotation is based on
phylogenetically distant taxa. Furthermore, while palaeogenomics
can provide evidence for community changes over time, there is
rarely sufcient resolution for population-level analysis (although
this might change, see a recent perspective paper90), and it cannot
provide a direct link to specic changes at the phenotype level
and the organismstness. Resurrection studies can address these
limitations.
One of the benets of sed-eDNA comes from its ability to
provide information about organisms that may not leave an
obvious fossil morphological record (Figs. 1and 3). However, as
with the development of other biochemical proxies in palaeoe-
cology, for example, pigments and stable isotopes, without a
critical context, sed-eDNA results can simply be another strati-
graphic prole to be interpreted subjectively. A more sophisti-
cated approach is to use the molecular results to answer questions
that are difcult to address with more traditional palaeoecological
approaches. Increased nutrient loading to aquatic ecosystems
results in greater production and changed species composition
and community structure91. However, high temporal resolution
stratigraphic sequences from eutrophic lakes show considerable
temporal variability in diatom species and cyanobacteria. Using a
multi-proxy approach with relevant statistical methods, it is clear
that increasing nutrient load, grazing by zooplankton or climate
forcing cannot explain all of this temporal variability in algal
abundance. In this context, sed-eDNA can provide supplemen-
tary evidence of other trophic links. For example, the role of
chytrid infections in controlling phytoplankton abundance has
been underestimated in freshwaters (see review by Frenken
et al.92). From a longer temporal perspective, combining fossil
Asterionella abundance with the sed-eDNA record of the chytrid
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8COMMUNICATIONS BIOLOGY | (2020) 3:169 | https://doi.org/10.1038/s42003-020-0899-z | www.nature.com/commsbio
Zygorhizidium planktocnium could provide evidence of an
important mechanism controlling Asterionella abundance at
decadal time scales. See also the work of Kyle85 discussed above.
A combination of resurrection ecology and sed-eDNA could also
be used to test the effect of environmental change on biodiversity,
e.g. how genetic diversity changes when one member disappears,
and to identify possible corresponding phenotypic responses in
the remaining species.
Conclusions: tracing evolution of planktonic food-webs in
sediment archives
The science of palaeoecology allows us to reconstruct past
changes in biological communities, but has limitations in species
coverage and in the type of information that can be inferred about
past processes. New developments in resurrection ecology and
sedimentary timeseries of eDNA promise to address these gaps
and can become powerful tools for predicting futures changes to
ecosystem function. Building on our knowledge about preserva-
tion and degradation of organic molecules in aquatic sediments,
we can develop and optimise sampling and interpretation of these
unique historical and ancient continuous time series of genetic
and phenotypic adaptation. DNA preserved in dated sediment
cores has the potential to increase taxonomic coverage to include
key organisms and processes that are missing in the microfossil
record. Resurrection ecology further adds the dimension of
linking population genetic changes with adaptive trait shifts, and
linking both to environmental drivers. Moreover, expanding
coverage in terms of species, organism groups and strain numbers
will allow us to reconstruct rates and magnitudes of change under
both natural and anthropogenic forcing.
We are condent that in a few years we will be able to address,
for example, questions about the sources of adaptive variation
and their underlying genomic architecture. Using undisturbed
aquatic sediment cores is the only way to obtain such continuous
records for high-resolution temporal reconstructions. The quan-
titative data acquired from this approach thus have the potential
to illuminate generic adaptive processes and responses in the face
of multiple environmental stressors.
Received: 22 October 2019; Accepted: 10 March 2020;
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Author contributions
All authors were participants in a workshop at Loughborough University in March 2017,
led by M.E. and N.J.A., and aimed at debating new possibilities and challenges in using
genetic signals stored in aquatic sediment archives as tools in evolutionary ecology. M.E.,
M.C., T.C., D.F., A.G., A.K., A.L., T.M., S.R., and N.J.A. have subsequently contributed to
writing this position paper.
Competing interests
The authors declare no competing interests.
Additional information
Correspondence and requests for materials should be addressed to M.E.
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... Possibly, the smaller DNA fragments in our samples were more susceptible to degradation than longer ones or were too short to pass our bioinformatics filtering criteria (all fragments <25 bp were removed during data processing); however, this is speculative and only considers the eukaryotic sequences rather than the whole sample. Fragment size is a relatively simple measure for the authenticity of sedaDNA as many factors can influence fragmentation, including chemical and physical properties, taphonomy, and diagenesis (Ellegaard et al., 2020;Giguet-Covex et al., 2019). In ancient bone samples, sample age does not necessarily correlate with increased fragmentation; however, sample age does correlate with increased DNA deamination frequency (Kistler et al., 2017), thus the latter is a more robust approach to assess DNA authenticity. ...
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... Elevated organic matter (OM) content seems to play a crucial role in trapping DNAses to soil colloids and minerals and hence, reducing the degradation speed of exDNA (Cai et al., 2006). In aquatic environments, UV-radiation, dissolved OM-and salt concentrations are additional constraints potentially influencing exDNA decay (Ellegaard et al., 2020;Zhang et al., 2020). In addition, also the bind- Microbial hotspots such as the rhizosphere or bioreactors, but also soils and sediments with high microbial turnover and low clay F I G U R E 2 Recovery and extraction of different DNA types from environmental samples. ...
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Human impacts on biodiversity are well recognized, but uncertainties remain regarding patterns of diversity change at different spatial and temporal scales. Changes in microbial assemblages are, in particular, not well understood, partly due to the lack of community composition data over relevant scales of space and time. Here, we investigate biodiversity patterns in cyanobacterial assemblages over one century of eutrophication and climate change by sequencing DNA preserved in the sediments of ten European peri-Alpine lakes. We found species losses and gains at the lake scale, while species richness increased at the regional scale over approximately the past 100 years. Our data show a clear signal for beta diversity loss, with the composition and phylogenetic structure of assemblages becoming more similar across sites in the most recent decades, as have the general environmental conditions in and around the lakes. We attribute patterns of change in community composition to raised temperatures affecting the strength of the thermal stratification and, as a consequence, nutrient fluctuations, which favoured cyanobacterial taxa able to regulate buoyancy. Our results reinforce previous reports of human-induced homogenization of natural communities and reveal how potentially toxic and bloom-forming cyanobacteria have widened their geographic distribution in the European temperate region.
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