Centennial clonal stability of asexual Daphnia in Greenland lakes despite climate variability


Climate and environmental condition drive biodiversity at many levels of biological organization, from populations to ecosystems. Combined with paleoecological reconstructions, palaeogenetic information on resident populations provides novel insights into evolutionary trajectories and genetic diversity driven by environmental variability. While temporal observations of changing genetic structure are often made of sexual populations, little is known about how environmental change affects the long-term fate of asexual lineages. Here, we provide information on obligately asexual, triploid Daphnia populations from three Arctic lakes in West Greenland through the past 200-300 years to test the impact of environmental change on the temporal and spatial population genetic structure. The contrasting ecological state of the lakes, specifically regarding salinity and habitat structure may explain the observed lake-specific clonal composition over time. Palaeolimnological reconstructions show considerable regional environmental fluctuations since 1,700 (the end of the Little Ice Age), but the population genetic structure in two lakes was almost unchanged with at most two clones per time period. Their local populations were strongly dominated by a single clone that has persisted for 250-300 years. We discuss possible explanations for the apparent population genetic stability: (a) persistent clones are general-purpose genotypes that thrive under broad environmental conditions, (b) clonal lineages evolved subtle genotypic differences unresolved by microsatellite markers, or (c) epigenetic modifications allow for clonal adaptation to changing environmental conditions. Our results motivate research into the mechanisms of adaptation in these populations, as well as their evolutionary fate in the light of accelerating climate change in the polar regions.
Ecology and Evolution. 2020;00:1–11.
Received: 4 September 2020 
  Revised: 12 October 2020 
  Accepted: 14 October 2020
DOI: 10.1002/ece3.7012
Centennial clonal stability of asexual Daphnia in Greenland
lakes despite climate variability
Maison Dane1| Nicholas John Anderson2| Christopher L. Osburn3|
John K. Colbourne1| Dagmar Frisch1
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
© 2020 The Authors. Ecolog y and Evolution published by John Wiley & Sons Ltd.
1School of Biosciences, University of
Birmingham, Birmingham, UK
2Department of Geography, Loughborough
University, Loughborough, UK
3Department of Marine, Earth, and
Atmospheric Sciences, North Carolina State
University, Raleigh, NC , USA
Dagmar Frisch, School of Biosciences,
University of Birmingham, Birmingham, UK.
Funding information
DF received funding from the European
Union's Horizon 2020 research and
innovation program under the Marie
Skłodowska-Curie grant agreement No.
658714 .
Climate and environmental condition drive biodiversity at many levels of biologi-
cal organization, from populations to ecosystems. Combined with paleoecological
reconstructions, palaeogenetic information on resident populations provides novel
insights into evolutionary trajectories and genetic diversity driven by environmen-
tal variability. While temporal observations of changing genetic structure are often
made of sexual populations, little is known about how environmental change affects
the long-term fate of asexual lineages. Here, we provide information on obligately
asexual, triploid Daphnia populations from three Arctic lakes in West Greenland
through the past 200–300 years to test the impact of environmental change on the
temporal and spatial population genetic structure. The contrasting ecological state
of the lakes, specifically regarding salinity and habitat structure may explain the ob-
served lake-specific clonal composition over time. Palaeolimnological reconstruc-
tions show considerable regional environmental fluctuations since 1,700 (the end
of the Little Ice Age), but the population genetic structure in two lakes was almost
unchanged with at most two clones per time period. Their local populations were
strongly dominated by a single clone that has persisted for 250–300 years. We dis-
cuss possible explanations for the apparent population genetic stability: (a) persis-
tent clones are general-purpose genotypes that thrive under broad environmental
conditions, (b) clonal lineages evolved subtle genotypic differences unresolved by
microsatellite markers, or (c) epigenetic modifications allow for clonal adaptation to
changing environmental conditions. Our results motivate research into the mecha-
nisms of adaptation in these populations, as well as their evolutionary fate in the light
of accelerating climate change in the polar regions.
asexual Daphnia, egg bank, Greenland, lake sediment, palaeogenetic, palaeolimnology
   DANE Et Al.
Climate and the environment are major drivers of biodiversity in the
broad sense. In addition to higher levels of biological organization
(e.g., communities and ecosystems), these environmental drivers
affect species at the population level, including their temporal and
spatial genetic structure and diversity (Pauls et al., 2013; Scheffers
et al., 2016). The responses of population genetic structure and di-
versity to environmental forcing need to be considered at a range
of temporal and spatial scales to fully evaluate how global change
processes affect species' distributions and adaptive capacities
(Ellegaard et al., 2020; Orsini et al., 2013). Long-term, field-based
perspectives that span pre- and postdisturbance time periods (typi-
cally >100 years to cover preindustrial times) are required to balance
experimental and laboratory approaches at elucidating ecological
responses and genetic adaptation to climate change and chemical
pollutants (Nogues-Bravo et al., 2018). Research that combines pa-
leoecological and molecular data was initiated in the late 1990s/
early 2000s (e.g., Kerfoot et al., 1999; Limburg & Weider, 2002;
Mergeay et al., 2006) and has since developed into an established
field to advance our understanding of past and current adaptation
to environmental shifts (reviewed in Ellegaard et al., 2020; Napier
et al., 2020).
Palaeolimnological approaches have a long history of providing
detailed reconstructions of ecosystem variability at a range of tem-
poral scales (100 to 103 years) and across trophic levels (Jeppesen
et al., 2017). Recent methodological progress combined with a bet-
ter understanding of preservational constraints and artifacts (e.g.,
seeds, eggs, cysts) has allowed the use of bulk (environmental) and
compound-specific (organismal) DNA to address questions about
long-term genetic trends, variability, and adaptation in aquatic eco-
systems (reviewed in Nogues-Bravo et al., 2018; Weider et al., 2018).
The application of DNA extracted from dormant propagules can also
be coupled with resurrection approaches (Burge et al., 2018; Weider
et al., 2018). In optimal conditions, the palaeogenetic record allows
the reconstruction of temporal patterns of genetic structure and
diversity of aquatic populations and communities. These palaeoge-
netic reconstructions can be coupled with more standard paleoeco-
logical proxies (microfossils, geochemical markers, etc.) that reflect
environmental and climatic drivers and forcing.
Genetic diversity is often reduced during times of environmental
disturbance following strong selection and resulting population bot-
tlenecks (Banks et al., 2013). Shifts in genetic structure associated
with rapid environmental change have also been observed in long-
term studies (Frisch et al., 2014; Lundholm et al., 2017). Likewise,
the spatial genetic structure of populations is strongly related to en-
vironmental gradients, creating patterns of isolation by environment
(Wang & Bradburd, 2014). Many species have undergone adaptive
evolution to shifts in environmental conditions (Carroll et al., 2007).
While these observations are generally made in sexual populations,
little is known about long-term dynamics in the genetic make-up
of asexual populations. Under an environmental change scenario,
asexual lineages may be at a disadvantage compared with sexuals,
who shuffle their genetic material thus creating the variation needed
to fuel selection (Crow, 1994; Lachapelle & Bell, 2012). However,
the potential of asexual lineages to persist during the rapid change
currently recorded across ecosystems remains unclear.
At higher latitudes, geographical parthenogenesis is prevalent in
various organisms, including Daphnia, and is often associated with
polyploidy (reviewed in Tilquin & Kokko, 2016). Obligate partheno-
genesis in the Daphnia pulex species complex involves the production
of apomictic eggs (Dufresne et al., 2011; Innes & Dufresne, 2020)
that are deposited in an ephippium. This chitinous structure is de-
rived from maternal tissue, and each contains two dormant eggs,
which in asexual populations are genetically identical. Ephippia with
unhatched eggs can be extracted from the sediment and their DNA
examined, allowing for a detailed study of temporal population ge-
netic structure of Daphnia populations (Frisch et al., 2016; Limburg
& Weider, 2002; Lundholm et al., 2017; Mergeay et al., 2007; Orsini
et al., 2016). Long-term studies at the scale of centuries on the fate
of asexual Daphnia lineages do not exist. However, various asex-
ual Daphnia lineages have been recorded for 20–30 years in the
Canadian Subarctic (Weider et al., 2010), and for over 70 years in
an African lake (Mergeay et al., 2006). Contrasting the idea of cen-
tennial persistence of asexual lineages is the age estimate of only
decades (Tucker et al., 2013) for asexual lineages of the North
American Daphnia pulex-complex that arose by contagious asexual-
ity (Paland et al., 2005).
Arctic lakes in Greenland provide an ideal system to study
long-term dynamics of asexual Daphnia populations in relation to
climate change and environmental forcing. Many lakes are fishless
and inhabited by populations of the keystone herbivore Daphnia,
in particular the large-bodied Daphnia pulex-complex (Jeppesen
et al., 2017). Arctic Daphnia populations are generally obligate asex-
uals (Decaestecker et al., 2009; Weider et al., 1996), and many of
these lineages are triploids (Vergilino et al., 2009). In the Illulissat
area of West Greenland, clonal richness of Daphnia pulicaria s.l. pop-
ulations was between 4 and 5 genotypes (Haileselasie, Mergeay,
Weider, Jeppesen, et al., 2016; Haileselasie, Mergeay, Weider,
Sommaruga, et al., 2016). The lakes along Kangerlussuaq in SW
Greenland are particularly well studied in relationship to recent cli-
mate change, with evidence for a dramatic increase in environmental
forcing of these sensitive habitats (Saros et al., 2019). In the context
of the present study, the Kangerlussuaq area is a natural experimen-
tal site, in that direct anthropogenic impacts are limited or absent.
But there have been substantial environmental changes reflecting
climate (temperature, precipitation, and altered seasonality) as well
as related landscape changes including soil erosion, dust defla-
tion, lake shrinkage, and terrestrial vegetation changes (Anderson
et al., 2017). Kangerlussuaq, in contrast to most Arctic regions, was
cooling throughout much of the 20th century. However, since 1996
the area has undergone pronounced climate change, including rapid
warming (>2°C) over the last 15 years, partly in relation to changes
in the Greenland Blocking Index (Saros et al., 2019).
DANE Et Al .
In order to advan ce our und erst anding of the fate of ase xua l pop -
ulations in the light of current environmental change at high latitudes,
there is a need for empirical data, especially on decadal to centen-
nial timescales. Here, we examine genetic diversity in polymorphic
microsatellite DNA, and spatio-temporal genetic structure of three
Daphnia lake populations in West Greenland near Kangerlussuaq
and their relationship with the known regional environmental his-
tory. We address the question if rapid environmental change over
the last 200–300 years has affected temporal and spatial population
genetic structure of asexual Daphnia populations at the individual
lake and landscape scale.
2.1 | Study area
The Kangerlussuaq area of SW Greenland is a major lake district
with thousands of lakes, with a reasonably well understood re-
gional limnology (Whiteford et al., 2016). This, coupled with pre-
liminary zooplankton surveys and the fact that a number of the
lakes have been the subject of palaeolimnological analyses (e.g.,
Law et al., 2015; McGowan et al., 2008) meant that sites could
be chosen to optimize ephippia recovery, namely deep season-
ally anoxic basins. Braya Sø (SS4) was also known to have high
concentrations of sediment biomarkers (D'Andrea et al., 2006).
The area around the head of the fjord (Figure 1) has low effec-
tive precipitation and is characterized by limited hydrological
connectivity resulting in a number of closed-basin oligosaline
lakes (conductivity range 1,000–4,000 μS/cm). In general, the
Kangerlussuaq freshwater lakes at the head of the fjord have con-
ductivities around 200–500 μS/cm and DOC concentrations ca.
30–50 mg/L; DOC in the oligosaline lakes can be much higher
(80–100 mg/L (Anderson & Stedmon, 2007); nutrient and major
ion water chemistry is given in Table S1). The study lakes are lo-
cat e d wi t hin a few km of ea c h ot her (Figur e 1). SS13 81 an d SS159 0
are small scour basins (21.5 and 24.6 ha, respectively) with maxi-
mum depths around 18 m. Both lakes stratify strongly with an-
oxic hypolimnia. Braya is a larger, meromictic oligosaline lake
(73 ha). The ice-free period is from late-May/ early-June to late
September. The lakes can be considered pristine in comparison
with temperate systems in North America and NW Europe and
have no direct cultural impact apart from low levels of atmos-
pheric pollution (Bindler et al., 2001). The catchment vegetation
of all lakes is dwarf shrub tundra.
The study lakes are presently fishless, which is regarded as
a requirement for populations of the large-bodied Daphnia spe-
ciesto persist (Lauridsen et al., 2001), although reports of co-oc-
currence exist (Haileselasie, Mergeay, Weider, Sommaruga,
et al., 2016). All the lakes have variable water levels both at in-
terannual and decadal timescales, resulting from their sensitiv-
ity to the seasonality of precipitation, the extent of the spring
melt input and intense evaporation during the summer (Anderson
et al., 20 01). Although the lakes are isolate d bas ins today, SS4 was
once part of a group of lakes that formed a large palaeolake in the
early Holocene (Aebly & Fritz, 2009); SS1590 would have drained
into this larger lake for a brief period. SS1381 is located 6 km to
the west of SS1590 and above the level of the highest shoreline
of the palaeolake.
West Greenland has undergone considerable environmental
change (over different timescales) since deglaciation. Research has
focussed on a range of biophysical aspects of the ice-land-water
continuum, including glacier mass balance, terrestrial vegetation
and herbivore dynamics, and aquatic biogeochemistry (Anderson
et al., 2017). Lake sediment records have provided details of ecolog-
ical response to climate and landscape change over centennial and
millennial timescales, including diatom, pigment, and palaeoclimatic
reconstructions (Law et al., 2015; McGowan et al., 2003, 2008;
Presthus Heggen et al., 2010). Because ephippia recovery was great-
est at SS4 and this lake has a well-defined environmental history,
discussion of microsatellite variability and environmental history is
restricted to this site.
FIGURE 1 Overview of the study area.
Insert shows location of the study lakes
SS4 (Braya Sø), SS1590, and SS1381
   DANE Et Al.
2.2 | Sediment sampling and analysis
Short sediment cores (4 replicates per lake) were taken in July 2015
with a 9.5 cm diameter Hon-Kajak sediment corer from the deepest
part of each lake. Replicate cores were taken at one location in the
basin, approximately within a 30-m radius. The boat was moved after
each core was taken to avoid unnecessary sediment disturbance.
Recovery was around 25-cm at each lake and at all sites included an
intact sediment water interface (Figure S1). Cores were sectioned on
shore at 1-cm intervals and samples were placed in individual plastic
bags. Although sedimentation rates are low in many of these lakes
(~0.03 cm/yr; Anderson et al., 2019), a 1-cm interval was used to maxi-
mize recovery of ephippia; finer interval sampling would have provided
too few ephippia. Samples were kept refrigerated after return from the
field until fur ther analysis. A subsample of each core slice was analyzed
for dry weight and organic matter content using standard methods
(loss-on-ignition at 55C). The cores used in this study were not dated
radiometrically but previous cores have been dated using 210Pb and 14C
(D'Andrea et al., 2011; McGowan et al., 2003). Organic matter profiles
are distinctive at each lake (Figure S2), allowing among-core correlation
and thus chronologies to be transferred from the dated cores to those
used in this study. This method is illustrated for SS4 where 4 replicate
cores were taken in 2015; core OM profiles are clearly repeatable
(Figure S2). Samples used for microsatellite analysis cover the most
recent 200–300 years for SS4 and SS1381. The higher sedimenta-
tion rate at SS1590 meant that recover y of ephippia with eggs suitable
for microsatellite analysis was confined to the most recent ~30 years
based on 210Pb analysis (NJ Anderson unpublished).
Previously, the changing abundance of purple sulfur bacteria (PSB)
at Braya Sø has been inferred from the variable concentration of the
carotenoid okenone in the lake sediment (see McGowan et al., 2008).
Unfortunately, pigments were not measured on the sediment cores
used in this study but we have shown a good agreement between
sediment porewater fluorescence of the cores sampled in 2015 and
okenone abundance from a 2001 core (Osburn & Anderson unpub-
lished). Therefore, in this study, we used the second component (C2)
of a PARAFAC model based on the fluorescence excitation-emission
matrices (EEMs) of porewater chromophoric dissolved organic matter
(CDOM) as a proxy for changing PSB abundance. Component 2 exhib-
its a clear peak centered on 280 nm and is a protein-like fluorescence
marker associated with bacteria. Porewater CDOM fluorescence was
measured on separate Varian Eclipse spectrofluorometers and fluores-
cence intensity was calibrated into Raman units.
The organic C flux was calculated as in previous studies
(Anderson et al., 2019), and at SS4 the ephippia accumulation rate
was estimated as the total number of ephippia in a 1-cm sediment
slice divided by the linear sediment accumulation rate (years/cm).
2.3 | Molecular analysis
The studied species was identified as Daphnia pulicaria sensu lato
(in the following: Daphnia pulicaria), and clustered with lineages of
the Polar Daphnia pulicaria clade based on the mitochondrial ND5
(Colbourne et al., 1998, D. Frisch, unpublished data).
Ephippia were removed from 1-cm-sections of sediment and
enumerated for each sediment section as described in Frisch
et al. (2014). Preference was given to sampling ephippia from a sin-
gle core. However, due to limited numbers of intact eggs suitable for
genetic analysis, we had to supplement ephippia from replicate cores
in several cases. For details on the replicate cores used see Table S3.
Eggs were removed from the ephippia and used for DNA extraction
(using one egg per ephippium). DNA was isolated from individual
eggs with the QIAamp Microkit (Qiagen Inc) following the protocol
for DNA extraction from tissue specified in the manufacturer's man-
ual. To facilitate DNA extraction, eggs were perforated with a sterile
micropipette tip, breaking the membrane and exposing egg contents
to the extraction buffers. The total number of DNA isolates was 92
from three lakes (n = 57 (SS4), n = 12 (SS1590), n = 23 (SS1381)).
Microsatellite loci were amplified in single, 12.5 µl multiplex
reactions (Type-it PCR kit, Qiagen Inc), using an Eppendorf Nexus
Thermal Cycler with thermal cycle conditions recommended in the
Type-it PCR kit manual. Ten microsatellite primers (Table S2) rep-
resenting genome-wide loci were used for genotyping; details in
Colbourne et al. (2004) and Frisch et al. (2014). Two primers (Dp90,
Dp377) failed to amplify in a consistent manner and were therefore
excluded from further analysis. Amplified microsatellites were geno-
typed on an Applied Biosystems 3730 genetic analyser. We used the
microsatellite plugin for Geneious 7.0.6 (https://www.genei
for peak calling and binning. Called peaks were visually inspected
and manually adjusted when necessary.
All analyses were run in R version 3.6.2 (R CoreTeam, 2017) using
the R package poppr 2.8.3 (Kamvar et al., 2014). For the population
genetic analyses, we excluded all isolates with >5% missing infor-
mation, yielding a set of 59 isolates (SS4: 28 isolates; SS1381: 10
isolates; SS1590: 21 isolates, Table S3). Analyses included the esti-
mation of allelic diversity for each locus, and the population genetic
parameters of estimated multilocus genotypes (eMLG) by rarefac-
tion to the smallest population size of 10 in SS1590.
Population genetic structure was visualized by a Multiple
Spanning Network computed with the R package poppr 2.8.3
(Kamvar et al., 2014). The MSN was constructed in poppr using
Bruvo's distance that takes into account stepwise mutational pro-
cesses (Bruvo et al., 2004).
3.1 | Environmental variability and ephippial
Regional climate in South West Greenland was variable from the
end of the Little Ice Age (LIA) with alternating warm and cold pe-
riods, and the most recent warm period beginning at the end of
the 20th century (Figure 2a). As example for the in-lake tempo-
ral variability in the last centuries, we focus on reconstruction of
DANE Et Al .
environmental conditions in SS4. Here, both the OC accumulation
rate and the abundance of PSB (as fluorescence PARAFAC compo-
nent C2) varied over the last 300 years suggesting variable lake pro-
duction (Figure 2b). Daphnia pulicaria ephippia flux varied from ca. 2
to 1,800 ephippia m2 yr−1 from 1600 AD to post 2000 AD, peaking
around 1900 AD (Figure 2c), indicative of considerable fluctuations
in population size.
3.2 | Population genetic parameters
Genetic diversity and population structure of Daphnia populations
were studied in three lakes near Kangerlussuaq, SW Greenland
(SS4, SS1381, SS1590, Figure 1), using dormant eggs deposited in
the sediment. The covered time period differed between lakes and
was 200–300 years in SS4 and SS1381, and ~30 years in SS1590.
All Daphnia clones were putatively triploid, based on the criterion
of three different alleles at a minimum of one locus in each multilo-
cus genotype (MLG). None of the MLGs included a locus with more
than three alleles. Of the eight loci used for genotyping, six (Dp162,
Dp291, Dp369, Dp401, Dp437, Dp461) had three different alleles in
at least one individual, while two (Dp43, Dp173) had a maximum of
two alleles.
Clonal diversity differed between the three lakes (Table 1): of 28
eggs examined in SS4, we found a total of three MLGs (maximum of
2 per time period, Figure 3a) of which one was dominant throughout
three centuries (MLG1). Only a single clone (MLG10) was detected in
the 21 isolates from SS1381, which persisted over several centuries,
as did MLG1 in SS4. In contrast, the Daphnia population in SS1590
was more diverse than the former two lakes with seven MLGs in a
total of 10 isolates, of which only one MLG was found during two
(nonadjacent) time periods. These differences in clonal diversity
and dominance structure are also reflected in the rarefied estimate
of clonal diversity (eMLG, Table 1), which in SS1590 was about 3.5
and 6 times higher than in SS4 and SS1381, respectively. Changes
in clonal composition over time were not discernible in either of the
two lakes from which long-term records across centuries could be
obtained (Figure 3a, SS4 and SS1381).
A Minimum Spanning Network shows the relationship between
the Daphnia lineages of the three lake populations (Figure 3b). Here,
the main genotype of SS4 (MLG1) was the most distant within the
network. It linked directly to the other two genotypes found in this
FIGURE 2 Environmental history
at SS4 (Braya Sø) and the response
ofDaphniagenotypes. (a) Climate
variability: reconstructed summer lake
water temperatures based on alkenones
at Braya Sø and Lake E (see (D'Andrea
et al., 2011) for details) and the mean
annual air temperature recorded at Nuuk.
(b) Aquatic production indices for the
lake: organic C burial reflects total in-lake
production and abundance of purple
sulfur bacteria inferred from porewater
fluorescence (as Parafac Component C2).
(c) The stability of the dominant genotype
(MLG1) over time period covered by the
sediment analyses is indicated by the solid
red line; two secondary, minor genotypes
(GT) are indicated (solid orange (MLG2)
and dotted red lines (MLG3)
   DANE Et Al.
location, suggesting MLG1 as the ancestral genotype in SS4. The
SS1590 genotypes formed two subnetworks, one of which was
linked directly with the only genotype detected in SS1381 (MLG10).
The results of this study indicated lake-specific genotypic composi-
tion of the three Daphnia populations among the lakes (Figure 3b),
and despite considerable in-lake and regional environmental change
since ~1700 AD (discussed below), the clonal structure of Daphnia
populations in two of three lakes (SS4 and SS1381) was remarkably
stable. Importantly, we found strong dominance of a single, lake-
specific asexual clone throughout the past two to three centuries in
each of these two lakes.
4.1 | Spatial patterns
The analysis of population genetic structure revealed a larger ge-
netic distance between the population of SS4 and the other two
lake populations (Figure 3b), a spatial pattern that may reflect the
primary environmental differences among the lakes as well as the
historic connectivity between these lakes: SS4 is oligosaline and
thus differs strongly from SS1381 and SS1590. The latter two
are quite similar to each other in terms of their water chemistry
(Table S1) and typical of many of the freshwater lakes around the
head of the fjord (Whiteford et al., 2016). Clearly, ionic composi-
tion and inorganic C chemistry is an important driver of genotypic
variability as much as it determines regional biodiversity and com-
munity structure (diatoms, chrysophytes (Pla & Anderson, 2005;
Ryves et al., 2002)). Conductivity is the dominant control on algal
composition in this area and is highly correlated with both DOC
concentration and CDOM quality (Anderson & Stedmon, 2007).
Conductivity was also identified as one of the major factors driv-
ing clonal composition of Daphnia populations in an area about
250 km north of Kangerlussuaq (Haileselasie, Mergeay, Weider,
Sommaruga, et al., 2016). Lakes SS1381 and SS1590 have never
been connected hydrologically, since SS1381 lies outside the
boundary of the large palaeolake that included SS4 in the period
immediately after deglaciation (Aebly & Fritz, 2009). The lack of
shared clones between lakes suggests local adaptation to the en-
vironmental conditions of each resident lake, in particular given
the geographic proximity between all three study lakes (less than
10 km) and the lack of evidence for dispersal limitation in other
populations of West Greenland Daphnia (Haileselasie, Mergeay,
Weider, Jeppesen, et al., 2016). However, because sample size was
limi ted , it is possible th at future sampling will dete c t additio nal , less
abundant genotypes that may be shared between locations.
The overall greater clonal diversity of the SS1590 Daphnia popu-
lat io n comp ared with that of SS4 and SS1381 may reflec t the greater
diversity of habitats in this lake—its two sub-basins are quite dif-
ferent in terms of their morphometry and substrate variability, in-
cluding the dominant macrophytes (Anderson, unpublished field
observations). Not only are littoral and benthic habitats more di-
verse in SS1590, but the lake also has a more dynamic response to
evaporative driven lake level changes than many of the lakes in the
area. These short-term lake level changes also exacerbate habitat
heterogeneity more at this site than others, due to its morphometry.
SS1381 has a simpler morphometry, essentially with one deep basin.
SS4, the largest of the lakes can be considered a pelagic system and
while lake levels are variable here as well, the contribution of the
littoral zone to whole lake production and diversity is probably more
4.2 | Regional environmental change
Lakes in the Kangerlussuaq area are tightly coupled to regional
climate change which influences both terrestrial landscape and
in-lake aquatic processes, such as hydrological runoff, terrestrial
productivity, lake levels, conductivity, and DOC concentration
(Osburn et al., 2019). The period covered by the genetic analyses
in two lakes, ~200–300 years, includes the end of the Little Ice
Age (LIA); a period of considerable change in regional climate, with
aridity and fluctuating temperatures. Lake levels would have been
lower than present (Aebly & Fritz, 2009; McGowan et al., 2003)
and aridity affects dust deflation from the sandurs immediately
north and to the east of the study area. As the lakes are strongly
P-limited (Brutemark et al., 2006), dust is a possible important nutri-
ent source. Atmospheric reactive N deposition has increased across
the Arctic (Wolfe et al., 2013) and Greenland is no exception. The
changing nutrient balance associated with these varying sources
will have impacted primary production and thus secondary produc-
ers (i.e., Daphnia). But as well as these broader, regional responses
which can be traced across lakes, each individual lake has its own
ecological trajectory in environmental space (see for example Law
et al., 2015). Long-term air temperature records show considerable
variability during the 20th century, including a period of pronounced
cooling. Rapid warming characterizes the start of the 21st century.
Lake temperatures track air temperatures tightly in this area (Kettle
et al., 2004) and so epilimnetic temperatures would have varied
TABLE 1 Genetic diversity of the Daphnia pulicaria population
in three lakes of West Greenland integrated over several sediment
SS4 28 31.7 0.7
SS159 0 10 66.0 0.0
SS1381 21 11.0 0.0
Tot al 59 10 3.9 1.1
Abbreviations: eMLG, number of expected MLG at the smallest sample
size based on rarefaction; MLG, Multilocus genotypes; N, number of
genotyped isolates; SE, Standard Error based on eMLG.
DANE Et Al .
As an example for local environmental variability since LIA,
SS4 showed pronounced variation of okenone, a pigment which
can be used as a proxy for purple sulfur bacteria (PSB) abundance
(McGowan et al., 2008). In SS4, in conjunction with indicators of
primary production, variation of ephippial densities provides ev-
idence of historic changes in trophic interactions. Ephippial pro-
duction serves as a rough estimate of Daphnia population size
(Jankowski & Straile, 2003; Nykänen et al., 2009). There is some
suggestion that over the last ~800 years the Daphnia population in
SS4 has been tracking purple sulfur bacteria (Frisch & Osburn un-
published), which Daphnia may exploit as a direct or indirect food
source, as obser ved in other studies (Jürgens et al., 1996; Massana
et al., 1994).
4.3 | Long-term persistence of dominant clones
Despite these profound environmental changes, there is limited gen-
otypic variability over time. We did not find evidence for environ-
mental dynamics to drive patterns of population genetic structure
or genetic diversity over the past several centuries: Our data sug-
gest the presence of a single clone in two of the study lakes, in SS4
(MLG1) and SS1381 (MLG10), across all sample depths, over the past
200–300 years. In SS4, two other clones were detected in two dif-
ferent time periods, both with very low abundance. In contrast, the
third lake (SS1590) had a much shorter temporal record but a higher
number of clones. While it is obvious that clonal diversity in this lake
overall was much higher, the temporal pattern of genetic structure
FIGURE 3 Spatial and temporal
population genetic structure of
theDaphniapopulations in three lakes.
To avoid an artificial increase of identical
genotypes, only one egg per ephippium
was used for microsatellite analysis. (a)
Abundance of multilocus genotypes
(MLG1 to MLG10) with information on
lake and sediment age from which eggs
were isolated. Each row represents a
lake: SS4 (4 time periods), SS1381 (4
time periods), and SS1590 (three time
periods). (b) Minimum Spanning Network
(MSN) of MLGs identified in the three
lakes and time periods. Thicker, darker
edges correspond to more closely related
genotypes. Numbering of MLGs is the
same as in (a)
(ca. 2010 AD;0-1cm)
(ca. 2000 AD;1-2cm)
(ca.1990 AD;2-3cm)
(ca. 1910 AD;1-2cm)
(ca. 1840 AD; 2-3cm)
(ca. 1770 AD, 3-4cm)
(ca. 2010AD; 0-1cm)
(ca. 2010 AD;0-1cm)
(ca.1990 AD; 1-2cm)
(ca. 1950 AD; 2-3cm)
(ca. 1770 AD;6-7cm)
0.0250.041 0.076 0.174 0.49
SS4 (ca. 2010AD)
SS4 (ca. 1990AD)
SS4 (ca.1950AD)
SS4 (ca. 1770AD)
SS1590 (ca. 2010AD)
SS1590 (ca. 2000AD)
SS1590 (ca 1990AD)
SS1381 (ca. 2010AD)
SS1381 (ca. 1910AD)
SS1381 (ca. 1840AD)
SS1381 (ca. 1770AD)
Distance (Bruvo)
   DANE Et Al.
and diver sity can not be ide nti fie d wit h cer tainty due to the low num -
bers of isolates; overall ephippial density in this lake was much lower
(personal observation) with a smaller amount of well-preserved eggs
suitable for genetic analyses.
There are several tenable explanations for the apparent domi-
nance and persistence of single clones in two lakes
(i) The persistent clone may be a general-purpose genotype that suc-
cessfully exploits a variety of historic environments that are not (yet)
within a harmful range
The invasion and dominance of a single, asexual Daphnia clone
have been observed in several lakes throughout a wide geographic
range. For example, an asexual Daphnia pulex clone invaded the
population in Lake Naivasha, Kenia in the 1920s, and then displaced
the local sexual, genetically diverse Daphnia population (Mergeay
et al., 2006). Successful invasion of the same clone was observed
throughout Africa in a wide range of aquatic habitats and environ-
mental conditions, testifying to the exceptional niche breadth of
this asexual clone. Similar observations have been made in Japan
(So et al., 2015), where four asexual Daphnia clones invaded a large
number of aquatic habitats across a wide geographical range and
ecological conditions. In contrast, Jose and Dufresne (2010) did not
observe a higher tolerance of asexual, polyploid clones compared
with diploid clones within the environmental range tested.
In two of our studied Arctic populations, the persistence of
Daphnia clones despite major temperature changes over the last
centuries in the study area may reflect the considerable in-lake tem-
perature gradient that Daphnia experience on a re gular basis . At SS4,
daily vertical migration to graze directly or indirectly on PSB and
POC (DOC) in the metalimnion at SS4 (a distance of some 8–12 m)
would expose animals to a temperature change of >10°C, consider-
ably more than the temperature change during the recent millennia.
Moreover, the annual temperature range in the epilimnion is also in
the order of 10°C (D'Andrea et al., 2011). Although only SS4 has a
metalimnetic PSB plate, all three study lakes have substantial gradi-
ents in DOC and POC, suggesting that they are utilizing a microbial
loop in the lakes. The three study lakes are seasonally anoxic with
hypolimnetic reductions in O2 during both summer and winter. It is
possible that these seasonal environmental changes and the vertical
O2 gradients exert greater physiological stress in Daphnia than the
stress associated with regional warming. Of course, this genetic sta-
sis may not continue should environmental change in SW Greenland
stay at a similar rate (Saros et al., 2019).
(ii) Undetected by the applied genetic resolution, a single MLG may com-
prise several distinct clonal lineages, each adapted to a specific his-
toric environment
The possibility of cryptic genetic variation must be considered
due to the limited resolution offered by microsatellite markers which
cannot fully account for possibly existing genome-wide variation.
For example, using 12 microsatellite loci, So et al. (2015) detected
only four MLGs in asexual Daphnia pulex that had invaded Japanese
lakes and ponds. Interestingly, these MLGs comprised 21 mitochon-
drial haplotypes, indicating a higher genotype diversity than that re-
solved by the microsatellite loci (So et al., 2015), a finding that was
later confirmed using whole-genome sequencing (Tian et al., 2019).
In particular, these authors concluded that the observed divergent
traits in these closely related asexual genotypes evolved without
recombination from an ancestral clone by a limited number of func-
tionally significant mutations (Tian et al., 2019).
(iii) Epigenetic modifications explain clonal adaptation and allow clones
to persist
There is increasing evidence that epigenetic mechanisms con-
tribute to evolution (Asselman et al., 2016; Becker et al., 2011) and
to adaptive responses related to climate and environmental change
(Alakärppä et al., 2018; Metzger & Schulte, 2018; Münzbergová
et al., 2019). In the absence of genomic variation, it is becoming in-
creasingly evident that changes in epigenetic profiles could allow
for rapid, heritable adaptations to environmental cues that pre-
cede the more slowly evolving changes in DNA sequences (Deakin
et al., 2014), that is, the classical Darwinian mutation accumula-
tion and selection concept. Epigenetic modifications would allow
persistence of asexual clones in the absence of recombination and
generation of genetic variation, because transgenerational inheri-
tance of DNA methylation is highly likely in Daphnia, whose gam-
etes are derived from almost fully matured tissue (Wojewodzic &
Beaton, 2017).
Our study examined the possible impact of environmental change
on the centuries-long persistence of asexual clones in two Daphnia
populations. While the interpretation of our results is limited by
sample size and genetic resolution, they are, to our best knowledge,
the first to report the population genetic structure of asexual, poly-
ploid Daphnia populations of the circumpolar Daphnia pulex-complex
across century timescales. In order to test the ecological implications
of these findings, laboratory experiments are needed in future stud-
ies. In particular, representatives of the current populations can be
used to test whether the dominant clones exhibit phenotypic plas-
ticity with a wide tolerance toward local conditions (temperature,
food, and salinity), or whether local (and temporal) adaptation to
different conditions can be observed. If possible, hatchlings of eggs
from older sediments should be included to compare phenotypic and
transcriptomic responses of ancient and contemporary isolates. In
addition, the role of epigenetic modifications should be considered
in further studies (Frisch et al., 2014, 2020).
Other factors in addition to environmental change, includ-
ing predators, parasites, or pathogens could be additional drivers
of clonal composition and stability. Future, more intensive sam-
pling should be performed to reveal additional clonal lineages and
DANE Et Al .
potential interaction with the biotic environment; however, the ob-
served dominance patterns and persistence of individual clonal lin-
eages are unlikely to change. Our results stimulate several questions
relating to the mechanisms of adaptation in these populations, as
well as their evolutionary fate during the next decades and beyond,
on the assumption that climate change in Greenland and other Arctic
regions proceeds on the predicted trajectory with severe ecological
We are grateful to Erika Whiteford, Madeleine Giles, Amanda
Burson, and Tania Cresswell-Maynard for help with fieldwork, to
Chri s O'G rady for assista nce in th e lab ora tor y, to Andy Moss fo r pro-
viding laboratory space for core processing at UoB and to Fengjuan
Xiao (Lboro) for the loss-on-ignition analyses.
None declared.
Maison Dane: Formal analysis (equal); writing-original draft (sup-
porting). Nicholas John Anderson: Formal analysis (equal); visuali-
zation (equal); writing-original draft (equal). Christopher L . Osburn:
Formal analysis (supporting); writing-original draft (supporting).
John K. Colbourne: Formal analysis (supporting); writing-original
draft (supporting). Dagmar Frisch: Conceptualization (lead); formal
analysis (lead); funding acquisition (lead); investigation (lead); super-
vision (lead); visualization (equal); writing-original draft (equal).
Data that support the findings of this study (microsatellite scores;
SS4 ephippia counts, loss-on-ignition data, and Parafac compo-
nent C2 scores) are openly available from Zenodo at https://doi.
Nicholas John Anderson https://orcid.
Christopher L. Osburn
John K. Colbourne
Dagmar Frisch
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... We can extract DNA from these preserved diapausing eggs, and thereby reconstruct population genetic dynamics (Burge et al., 2018;Orsini et al., 2016). Although previous studies have revealed the evolutionary processes of Daphnia using diapausing eggs (e.g., Decaestecker et al., 2007;Hairston et al., 1999;Stoks et al., 2016), only three prior studies have observed long-term genetic dynamics of Daphnia species including the early stage of colonisation (Conde-Porcuna et al., 2021;Dane et al., 2020;Möst et al., 2015). Möst et al. (2015) analysed 40 years of long-term genetic dynamics of colonisation and establishment by Daphnia pulicaria that consisted of cyclic parthenogenetic lineages and possibly a few obligate parthenogenetic lineages. ...
... Möst et al. (2015) analysed 40 years of long-term genetic dynamics of colonisation and establishment by Daphnia pulicaria that consisted of cyclic parthenogenetic lineages and possibly a few obligate parthenogenetic lineages. Dane et al. (2020) and Conde-Porcuna et al. (2021) analysed long-term genetic dynamics of an obligate parthenogenetic Daphnia pulex species complex. Dane et al. (2020) observed the population genetic dynamics of obligate parthenogenetic D. pulicaria for three centuries including colonisation of three lakes in Greenland. ...
... Dane et al. (2020) and Conde-Porcuna et al. (2021) analysed long-term genetic dynamics of an obligate parthenogenetic Daphnia pulex species complex. Dane et al. (2020) observed the population genetic dynamics of obligate parthenogenetic D. pulicaria for three centuries including colonisation of three lakes in Greenland. Conde-Porcuna et al. (2021) observed the population genetic dynamics of obligate parthenogenetic D. pulex, which belonged to the North American lineage, for 65 years in a Spanish high-mountain lake. ...
Population genetic diversity and structure are known to influence successful colonisation and establishment of a population in a novel habitat. However, our understanding of actual population genetic changes in the early stage of colonisation and detailed time‐series of genetic changes during the establishment of a new population is still limited due to the difficulty of making long‐term direct observations of a colonising population. Using a paleolimnological approach, we revealed the long‐term genetic dynamics of an obligate parthenogenetic panarctic Daphnia pulex population during the early stage of colonisation and subsequent establishment in Lake Fukami‐ike of Nagano, Japan. We collected diapausing eggs in ephippia of D. pulex from layers of sediment core samples (c. 370 cm long; dated 1662–2017) and then the mitochondrial DNA was sequenced using the markers designed for the NADH dehydrogenase 5 (ND5) gene and the control region. Daphnia pulex colonised and established a population in the early 2000s. Population genetic analysis showed that one colonising haplotype predominated in the population throughout the study period. Genetic diversity was consistently limited and there was no recovery of genetic diversity after colonisation. Clearly limited genetic diversity did not hamper population growth, possibly because phenotypic plasticity was used to cope with changes in predation pressure. Analysis using multivariate auto‐regressive models showed that responses to environmental and ecological factors varied depending on the haplotype, which may have contributed to successful colonisation of the lake by the different haplotypes. The present results suggest that an obligate parthenogenetic population can establish in a new habitat and be maintained for generations even though it has limited genetic diversity. We found that two independent lineages (JPN1 and JPN2) successfully established and coexisted toward the end of the study period. Although limited genetic diversity did not impede colonisation success, how the later colonising JPN1 lineage has successfully invaded a population consisting solely of the early colonising JPN2 deserves future attention to understand the population genetic dynamics revealed in this study.
... The genus is most closely related to the Moinidae within the Cladocera (Schwentner et al. 2018) and consists of three subgenera (Australodaphnia, Ctenodaphnia, and Daphnia; Colbourne et al. 2006). The total number of accepted species in the genus was 87 in 2015 (Kotov 2015), but several species have been newly or re-described since (Zuykova et al. 2018(Zuykova et al. , 2019Garibian and Kotov 2020;Kotov et al. 2020 (Hamrová et al. 2010(Hamrová et al. , 2011Tucker et al. 2013;Dane et al. 2020), most populations undergo sexual reproduction at least once a year (Lampert 2011). The result of sexual reproduction is a resting egg in a chitinous case, called ephippium, from greek epi and híppos meaning "on the horse", because of the saddle-like shape and brown melanin-based colour. ...
... In response to massive input of phosphorus (Schindler 1974) As facultative parthenogenetic species, i.e. reproducing asexually throughout most of the year some, Daphnia has several ways to resist hybridization and introgression. While most populations undergo sexual reproduction once a year, some populations can survive for decades without sexual reproduction (Tucker et al. 2013;Dane et al. 2020), thereby being resistant to even long-term hybridization. Alternatively, sexually reproduced dormant eggs encased in protective ephippia, that allow Daphnia to overwinter, may persist up to several years to centuries, before hatching (Hairston et al. 1995;Frisch et al. 2014). ...
... However, this would ignore the strong differentiation between many species within this complex (Chapters IV and V) and the fact that many species exclusively produce hybrids with D. galeata sensu lato and D. cucullata sensu lato (Schwenk and Spaak 1997;Schwenk et al. 2000). This leads to the question of how clones can be classified, if they have reproduced asexually for decades, such as the hybrids found in Lake Constance (Chapter II) or, similarly, obligate parthenogenetic clones in the arctic (Tucker et al. 2013;Dane et al. 2020 ...
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Several genetic lineages of obligate parthenogenetic Daphnia pulex, a common zooplankton species, have invaded Japan from North America. Among these, a lineage named JPN1 is thought to have started colonization as a single genotype several hundred to thousand years ago and subsequently produced many genotypes in Japan. To examine the phenotypic variations due to ecological drivers diverging the genotypes in new habitats, we measured heritability and variation in 17 traits, including life history, morphology and digestive traits, and the genetic distance among the D. pulex JPN1 genotypes in Japan. We found that most of the traits measured varied significantly among the genotypes and that heritability was highest in the morphological traits, followed by the digestive and life history traits. In addition, 93% of the variation in these traits was explained by the first three components in the principal component analysis, implying that variation of these heritable traits is not random but rather converged into a few directions. These relations among traits revealed the potential importance of predation pressures and food conditions as factors for diverging and selecting different genotypes. However, the magnitude of the difference in any single trait group did not correlate with the genetic distance. Our findings show that the divergent traits evolved within D. pulex JPN1 lineage without genetic recombination, since their ancestral clone invaded Japan. Large variations and covariations of the phenotypic traits, irrespective of the genetic distance among the genotypes, support the view that the invasive success of D. pulex JPN1 was promoted by a genetic architecture that allowed for large phenotypic variations with a limited number of functionally important mutations without recombination.
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Lakes are a key feature of arctic landscapes and can be an important component of regional organic carbon (OC) budgets, but C burial rates are not well estimated. 210Pb-dated sediment cores and carbon and organic matter (as loss-on-ignition) content were used to estimate OC burial for 16 lakes in SW Greenland. Burial rates were corrected for sediment focusing using the 210Pb flux method. The study lakes span a range of water chemistries (conductivity range 25–3400 µS cm−1), areas (< 4–100 ha) and maximum depths (~ 10–50 m). The regional average focusing-corrected OC accumulation rate was ~ 2 g C m−2 y−1 prior to ~ 1950 and 3.6 g C m−2 y−1 after 1950. Among-lake variability in post-1950 OC AR was correlated with in-lake dissolved organic carbon concentration, conductivity, altitude and location along the fjord. Twelve lakes showed an increase in mean OC AR over the analyzed time period, ~ 1880–2000; as the study area was cooling until recently, this increase is probably attributable to other global change processes, for example, altered inputs of N or P. There are ~ 20,000 lakes in the study area ranging from ~ 1 ha to more than 130 km2, although over 83% of lakes are less than 10 ha. Extrapolating the mean post-1950 OC AR (3.6 g C m−2 y−1) to all lakes larger than 1000 ha and applying a lower rate of ~ 2 g C m−2 y−1 to large lakes (> 1000 ha) suggests a regional annual lake OC burial rate of ~ 10.14 × 109 g C y−1 post 1950. Given the low C content of soils in this area, lakes represent a substantial regional C store.
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Environmentally induced epigenetic variation has been recently recognized as a possible mechanism allowing plants to rapidly adapt to novel conditions. Despite increasing evidence on the topic, little is known on how epigenetic variation affects responses of natural populations to changing climate. We studied the effects of experimental demethylation (DNA methylation is an important mediator of heritable control of gene expression) on performance of a clonal grass, Festuca rubra, coming from localities with contrasting temperature and moisture regimes. We compared performance of demethylated and control plants from different populations under two contrasting climatic scenarios and explored whether the response to demethylation depended on genetic relatedness of the plants. Demethylation significantly affected plant performance. Its effects interacted with population of origin and partly with conditions of cultivation. The effects of demethylation also varied between distinct genotypes with more closely related genotypes showing more similar response to demethylation. For belowground biomass, demethylated plants showed signs of adaptation to drought that were not apparent in plants that were naturally methylated. The results suggest that DNA methylation may modify the response of this species to moisture. DNA methylation may thus affect the ability of clonal plants to adapt to novel climatic conditions. Whether this variation in DNA methylation may also occur under natural conditions, however, remains to be explored. Despite the significant interactions between population of origin and demethylation, our data do not provide clear evidence that DNA methylation enabled adaptation to different environments. In fact, we obtained stronger evidence of local adaptation in demethylated than in naturally‐methylated plants. As changes in DNA methylation may be quite dynamic, it is thus possible that epigenetic variation can mask plant adaptations to conditions of their origin due to pre‐cultivation of the plants under standardized conditions. This possibility should be considered in future experiments exploring plant adaptations. This article is protected by copyright. All rights reserved.
While the interplay between migration and adaptation dictates species response to climate change, technological limitations have obfuscated explicit tests on past adaptive responses. However, a surge in technology-driven advances in paleoecological methods coincides with breakthroughs in processing ancient DNA, providing the first opportunity to assess adaptation to past climate shifts.
Knowledge of the molecular basis of phenotypic responses to environmental cues is key to understanding the process of adaptation. Insights to adaptation at an evolutionary time scale can be gained by observing organismal responses before and after a shift in environmental conditions, but such observations can rarely be made. Using the ecological and genomic model Daphnia, we linked transcriptomic responses and phosphorus(P)-related phenotypic traits under high and low P availability. We mapped weighted gene co-expression networks to traits previously assessed in resurrected ancient (600 years-old) and modern Daphnia pulicaria from a lake with a historic shift in P-enrichment. Subsequently, we assessed evolutionary conservation or divergence in transcriptional networks of the same isolates. We discovered highly preserved gene networks shared between ancient genotypes and their modern descendants, but also detected clear evidence of transcriptional divergence between these evolutionarily separated genotypes. Our study highlights that phenotypic evolution is a result of molecular fine-tuning on different layers ranging from basic cellular responses to higher order phenotypes. In a broader context, these findings advance our understanding how populations are able to persist throughout major environmental shifts.
How individual species and entire ecosystems will respond to future climate change are among the most pressing questions facing ecologists. Past biodiversity dynamics recorded in the paleoecological archives show a broad array of responses, yet significant knowledge gaps remain. In particular, the relative roles of evolutionary adaptation, phenotypic plasticity, and dispersal in promoting survival during times of climate change have yet to be clarified. Investigating the paleo-archives offers great opportunities to understand biodiversity responses to future climate change. In this review we discuss the mechanisms by which biodiversity responds to environmental change, and identify gaps of knowledge on the role of range shifts and tolerance. We also outline approaches at the intersection of paleoecology, genomics, experiments, and predictive models that will elucidate the processes by which species have survived past climatic changes and enhance predictions of future changes in biological diversity.
Long-lived conifers are vulnerable to climate change because classical evolutionary processes are slow in developing adaptive responses. Therefore, the capacity of a genotype to adopt different phenotypes is important. Gene expression is the primary mechanism to convert genome-encoded information into phenotypes, and further, DNA methylation is employed in epigenetic regulation of gene expression. We investigated variation in global DNA methylation and gene expression between three Scots pine (Pinus sylvestris L.) populations located in northern and southern Finland using mature seeds. Gene expression levels were studied in six DNA methyltransferase (DNMT) genes, which were characterized in this study, and in 19 circadian clock genes regulating adaptive traits. In embryos, expression diversity was found from three DNMT genes, which maintain DNA methylation. Expression of two DNMT genes was strongly correlated with climate variables, which suggests a role for DNA methylation in local adaptation. In adaptation-related genes, expression levels showed among-population variation in 11 genes in megagametophytes and in eight genes in embryos, and many of these genes were linked to climate factors. Altogether, our results suggest that differential DNA methylation and gene expression contribute to local adaptation in Scots pine populations and may enhance the fitness of trees under rapidly changing climate.