1
Centennial clonal stability of asexual Daphnia in Greenland lakes despite climate 1
variability 2
3
4
Maison Dane1 5
N. John Anderson2 6
Christopher L. Osburn3 7
John K. Colbourne1 8
Dagmar Frisch1, * 9
10
1 School of Biosciences, University of Birmingham, UK 11
2 Department of Geography, Loughborough University, UK 12
3 Department of Marine, Earth, and Atmospheric Sciences, North Carolina State University, 13
USA 14
* corresponding author 15
16
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2
Abstract 17
Climate and environmental condition drive biodiversity at many levels of biological 18
organisation, from populations to ecosystems. Combined with palaeoecological 19
reconstructions, palaeogenetic information on resident populations provides novel insights 20
into evolutionary trajectories and genetic diversity driven by environmental variability. While 21
temporal observations of changing genetic structure are often made of sexual populations, 22
little is known about how environmental change affects the long-term fate of asexual lineages. 23
Here, we provide information on obligately asexual, triploid Daphnia populations from three 24
Arctic lakes in West Greenland through the past 200-300 years to test the impact of a 25
changing environment on the temporal and spatial population genetic structure. The 26
contrasting ecological state of the lakes, specifically regarding salinity and habitat structure 27
may explain the observed lake-specific clonal composition over time. Palaeolimnological 28
reconstructions show considerable environmental fluctuations since 1700 (the end of the 29
Little Ice Age), but the population genetic structure in two lakes was almost unchanged with 30
at most two clones per time period. Their local populations were strongly dominated by a 31
single clone that has persisted for 250-300 years. We discuss three possible explanations for 32
the apparent population genetic stability: (1) the persistent clones are general purpose 33
genotypes that thrive under broad environmental conditions, (2) clonal lineages evolved 34
subtle genotypic differences that are unresolved by microsatellite markers, or (3) epigenetic 35
modifications allow for clonal adaptation to changing environmental conditions. Our results 36
will motivate research into the mechanisms of adaptation in these populations, as well as their 37
evolutionary fate in the light of accelerating climate change in the polar regions. 38
39
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3
Introduction 40
Climate and the environment are major drivers of biodiversity in the broad sense. In addition 41
to higher levels of biological organisation (e.g. communities, ecosystems), these 42
environmental drivers affect species at the population level, including their temporal and 43
spatial genetic structure and diversity [1,2]. The responses of population genetic structure and 44
diversity to environmental forcing need to be considered at a range of temporal and spatial 45
scales to fully evaluate how global change processes affect species' distributions and adaptive 46
capacities [3,4]. Long-term, field-based perspectives that span pre- and post-disturbance time 47
periods (typically > 100 years to cover pre-industrial times) are required to balance 48
experimental and laboratory approaches at elucidating ecological responses and genetic 49
adaptation to climate change and chemical pollutants [5]. Studies that combine 50
palaeoecological data with molecular data are especially promising to advance our 51
understanding of past and current adaptation to environmental shifts [3,6]. 52
Palaeolimnological approaches have a long history of providing detailed reconstructions of 53
ecosystem variability at a range of temporal scales (100 to 103 yrs) and across trophic levels 54
[7]. Recent methodological progress combined with a better understanding of preservational 55
constraints and artefacts (e.g. seeds, eggs, cysts) has allowed the use of bulk (environmental) 56
and compound-specific (organismal) DNA to address questions about long-term genetic 57
trends, variability and adaptation in aquatic ecosystems [5,8]. The application of DNA 58
extracted from dormant propagules can also be coupled with resurrection approaches [8,9]. In 59
optimal conditions, the palaeogenetic record allows the reconstruction of temporal patterns of 60
genetic structure and diversity of aquatic populations and communities. These palaeogenetic 61
reconstructions can be coupled with more standard palaeoecological proxies (microfossils, 62
geochemical markers, etc.) that reflect environmental and climatic drivers and forcing. 63
Genetic diversity is often reduced during times of environmental disturbance following strong 64
selection and resulting population bottlenecks [10]. Shifts in genetic structure associated with 65
rapid environmental change have also been observed in long-term studies [11,12]. Likewise, 66
the spatial genetic structure of populations is strongly related to environmental gradients, 67
creating patterns of isolation by environment [13]. Many species have undergone adaptive 68
evolution to shifts in environmental conditions [14]. While these observations are generally 69
made in sexual populations, little is known about long-term dynamics in the genetic make-up 70
of asexual populations. Under an environmental change scenario, asexual lineages may be at a 71
disadvantage compared with sexuals, by shuffling their genetic material thus creating the 72
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variation needed to fuel selection [15,16]. However, the potential of asexual lineages to 73
persist during the rapid change currently recorded across ecosystems remains unclear. 74
Obligate parthenogenesis in the Daphnia pulex species complex involves the production of 75
apomictic eggs [17,18] that are deposited in an ephippium. This chitinous structure is derived 76
from maternal tissue, and each contains two dormant eggs, which in asexual populations are 77
genetically identical. Ephippia with unhatched eggs can be extracted from the sediment and 78
their DNA examined, allowing for a detailed study of temporal population genetic structure of 79
Daphnia populations [12,19–22]. Long-term studies at the scale of centuries on the fate of 80
asexual Daphnia lineages do not exist. However, various asexual Daphnia lineages have been 81
recorded for 20-30 years in the Canadian Subarctic [23], and for over 70 years in an African 82
lake [20]. Contrasting the idea of centennial persistence of asexual lineages is the age estimate 83
of only decades [24] for asexual lineages of the North American Daphnia pulex-complex that 84
arose by contagious asexuality [25]. 85
Arctic lakes in Greenland provide an ideal system to study long-term dynamics of asexual 86
populations. Many lakes are fishless and inhabited by populations of the keystone herbivore 87
Daphnia, in particular the large-bodied Daphnia pulex-complex [7]. Arctic Daphnia 88
populations are generally obligate asexuals [26,27], and many of these lineages are triploids 89
[28]. In the Illulissat area of West Greenland, clonal richness of Daphnia pulex populations 90
was between 4 and 5 genotypes [29,30]. The lakes along Kangerlussuaq in SW Greenland are 91
particularly well-studied in relationship to recent climate change, with evidence for a dramatic 92
increase in environmental forcing of these sensitive habitats [31]. In the context of the present 93
study, the Kangerlussuaq area is a natural experimental site, in that direct anthropogenic 94
impacts are limited or absent. But there have been substantial environmental changes 95
reflecting climate (temperature, precipitation and altered seasonality) as well as related 96
landscape changes including soil erosion, dust deflation, lake shrinkage, and terrestrial 97
vegetation changes [32]. Kangerlussuaq, in contrast to most Arctic regions, was cooling 98
throughout much of the 20th century. However, since 1996 the area has undergone 99
pronounced climate change, including rapid warming (> 2 °C) over the last 15 years, partly in 100
relation to changes in the Greenland Blocking Index [31]. 101
In order to advance our understanding of the fate of asexual populations in the light of current 102
environmental change at high latitudes, there is a need for empirical data, especially on 103
decadal to centennial timescales. Here, we examine genetic diversity in polymorphic 104
microsatellite DNA, and spatio-temporal genetic structure of three Daphnia lake populations 105
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5
in West Greenland near Kangerlussuaq and their relationship with the known regional 106
environmental history. We test the hypothesis that rapid environmental change over the last 107
200-300 years has driven change in temporal and spatial population genetic structure of 108
asexual Daphnia populations at the individual lake and landscape scale. 109
110
Material and Methods 111
Study area 112
The Kangerlussuaq area of SW Greenland is a major lake district with thousands of lakes, 113
with a reasonably well understood regional limnology [33]. This, coupled with preliminary 114
zooplankton surveys and the fact that a number of the lakes have been the subject of 115
palaeolimnological analyses (e.g. [34,35]) meant that sites could be chosen to optimize 116
ephippia recovery, namely deep seasonally anoxic basins. Braya Sø (SS4) was also known to 117
have high concentrations of sediment biomarkers [36]. The area around the head of the fjord 118
(Fig. 1) has low effective precipitation and is characterized by limited hydrological 119
connectivity resulting in a number of closed-basin oligosaline lakes (conductivity range 120
1000–4000 µS cm-1). In general, the Kangerlussuaq freshwater lakes at the head of the fjord 121
have conductivities around 200-500 µS cm-1 and DOC concentrations ca. 30-50 mg L–1; DOC 122
in the oligosaline lakes can be much higher (80-100 mg L-1 [37]; nutrient and major ion water 123
chemistry is given in Table S1). The study lakes are located within a few km of each other 124
(Fig. 1). SS1381 and SS1590 are small (21.5 and 24.6 ha respectively) scour basins with 125
maximum depths around 18 m. Both lakes stratify strongly with anoxic hypolimnia. Braya Sø 126
is a larger, meromictic oligosaline lake (73 ha). The ice-free period is from late-May / early-127
June to late September. The lakes can be considered pristine in comparison to temperate 128
systems in North America and NW Europe and have no direct cultural impact apart from low 129
levels of atmospheric pollution [38]. The catchment vegetation of all lakes is dwarf shrub 130
tundra. 131
The study lakes are presently fishless, which is required for Daphnia populations to persist 132
[39]. All the lakes have variable water levels both at inter-annual and decadal timescales, 133
resulting from their sensitivity to the seasonality of precipitation, the extent of the spring melt 134
input and intense evaporation during the summer [40]. Although the lakes are isolated basins 135
today, SS4 was once part of a group of lakes that formed a large palaeolake in the early 136
Holocene [41]; SS1590 would have drained into this larger lake for a brief period. SS1381 is 137
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6
located 6 km to the west of SS1590 and above the level of the highest shoreline of the 138
palaeolake. 139
West Greenland has undergone considerable environmental change (over different timescales) 140
since deglaciation. Research has focussed on a range of biophysical aspects of the ice-land-141
water continuum, including glacier mass balance, terrestrial vegetation and herbivore 142
dynamics, and aquatic biogeochemistry [32]. Lake sediment records have provided details of 143
ecological response to climate and landscape change over centennial and millennial 144
timescales, including diatom, pigment and palaeoclimatic reconstructions [34,35,42,43]. 145
Because ephippia recovery was greatest at SS4 and this lake has a well-defined environmental 146
history, discussion of microsatellite variability and environmental history is restricted to this 147
site. 148
149
Sediment sampling and analysis 150
Short sediment cores (4 replicates per lake) were taken in July 2015 with a 9.5 cm diameter 151
Hon-Kajak sediment corer from the deepest part of each lake. Recovery was around 25-cm at 152
each lake and at all sites included an intact sediment water interface (Fig. S1). Cores were 153
sectioned on shore at 1-cm intervals and samples were placed in individual plastic bags. 154
Although sedimentation rates are low in these lakes (~0.3 cm yr–1; refs), a 1-cm interval was 155
used to maximise recovery of ephippia; finer interval sampling would have provided too few 156
ephippia. Samples were kept refrigerated after return from the field until further analysis. A 157
sub-sample of each core slice was analysed for dry weight and organic matter content using 158
standard methods (loss-on-ignition at 550 °C). The cores used in this study were not dated 159
radiometrically but previous cores have been dated using 210Pb and 14C [43,44]. Organic 160
matter profiles are distinctive at each lake (Fig. S2), allowing among-core correlation and thus 161
chronologies to be transferred from the dated cores to those used in this study. This method is 162
illustrated for SS4 where 4 replicate cores were taken in 2015; core OM profiles are clearly 163
repeatable (Fig. S2). Samples used for microsatellite analysis cover the most recent 200–300 164
years for SS4 and SS1381. The higher sedimentation rate at SS1590 meant that recovery of 165
ephippia with eggs suitable for microsatellite analysis was confined to the most recent ~30 166
years based on 210Pb analysis (NJ Anderson unpublished). 167
Previously, the changing abundance of purple sulphur bacteria (PSB) at Braya Sø has been 168
inferred from the variable concentration of the carotenoid okenone in the lake sediment (see 169
[34]). Unfortunately, pigments were not measured on the sediment cores used in this study 170
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but we have shown a good agreement between sediment porewater fluorescence of the cores 171
sampled in 2015 and okenone abundance from a 2001 core (Osburn & Anderson 172
unpublished). Therefore, in this study we used the second component (C2) of a PARAFAC 173
model based on the fluorescence excitation emission matrices (EEMs) of porewater 174
chromophoric dissolved organic matter (CDOM) as a proxy for changing PSB abundance. 175
Component 2 exhibits a clear peak centred on 280 nm and is a protein-like fluorescence 176
marker associated with bacteria. Porewater CDOM fluorescence was measured on separate 177
Varian Eclipse spectrofluorometers and fluorescence intensity was calibrated into Raman 178
units. 179
The organic C flux was calculated as in previous studies [45] and at SS4 the ephippia 180
accumulation rate was estimated as the total number of ephippia in a 1-cm sediment slice 181
divided by the linear sediment accumulation rate (yrs cm–1). 182
183
Molecular analysis 184
The studied species was identified as Daphnia pulex sensu lato (in the following: Daphnia 185
pulex), and clustered with lineages of the Polar Daphnia pulicaria clade based on the 186
mitochondrial ND5 [46] (D. Frisch, unpublished data). 187
Ephippia were removed from 1-cm-sections of sediment and enumerated for each sediment 188
section as described in Frisch et al. [12]. Eggs were removed from the ephippia and used for 189
DNA extraction (using one egg per ephippium). DNA was isolated from individual eggs with 190
the QIAamp Microkit (Qiagen Inc, Valencia, CA, USA) following the protocol for DNA 191
extraction from tissue specified in the manufacturer’s manual. To facilitate DNA extraction, 192
eggs were perforated with a sterile micropipette tip, breaking the membrane and exposing egg 193
contents to the extraction buffers. The total number of DNA isolates was 92 from three lakes 194
(n = 57 (SS4), n = 12 (SS1590), n = 23 (SS1381)). 195
Microsatellite loci were amplified in single, 12.5 µl multiplex reactions (Type-it PCR kit, 196
Qiagen Inc, Valencia, CA, USA), using an Eppendorf Nexus Thermal Cycler with thermal 197
cycle conditions recommended in the Type-it PCR kit manual. Ten microsatellite primers 198
(Table S2) representing genome-wide loci were used for genotyping; details in [12,47]). Two 199
primers (Dp90, Dp377) failed to amplify in a consistent manner and were therefore excluded 200
from further analysis. Amplified microsatellites were genotyped on an Applied Biosystems 201
3730 genetic analyser. We used the microsatellite plugin for Geneious 7.0.6 202
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(https://www.geneious.com) for peak calling and binning. Called peaks were visually 203
inspected and manually adjusted when necessary. 204
All analyses were run in R version 3.6.2 [48] using the R package poppr 2.8.3 [49]. For the 205
population genetic analyses, we excluded all isolates with >5% missing information, yielding 206
a set of 59 isolates (SS4: 28 isolates; SS1381: 10 isolates; SS1590: 21 isolates, Table S3). 207
Analyses included the estimation of allelic diversity for each locus, and the population genetic 208
parameters of estimated multilocus genotypes (eMLG) by rarefaction to the smallest 209
population size of 10 in SS1590. 210
Population genetic structure inferred by the number of clusters of genetically related 211
individuals was analysed using Discriminant Analysis of Principal Components (DAPC) 212
[50].This procedure involves the computation of a Principal Components Analysis (PCA) 213
followed by Discriminant Analysis (DA). The number of PCs to be used in DA was 214
identified by the function xvalDapc() implemented in poppr. 215
216
Results 217
Environmental variability and ephippial dynamics 218
Regional climate in South West Greenland was variable from the end of the Little Ice Age 219
(LIA) with alternating warm and cold periods, and the most recent warm period beginning at 220
the end of the 20th century (Fig. 2A). As example for the in-lake temporal variability in the 221
last centuries, we focus on reconstruction of environmental conditions in SS4. Here, both the 222
OC accumulation rate and the abundance of PSB (as fluorescence PARAFAC component C2) 223
varied over the last 300 years suggesting variable lake production (Fig. 2B). Daphnia pulex 224
ephippia flux varied from ca. 2 to 900 ephippia m2 yr-1 in 1700 to post 1950, peaking at 888 225
around 1900 (Fig. 2C), indicative of considerable fluctuations in population size. 226
227
Population genetic parameters 228
Genetic diversity and population structure of Daphnia populations were studied in three lakes 229
near Kangerlussuaq, SW Greenland (SS4, SS1381, SS1590, Fig. 1), using dormant eggs 230
deposited in the sediment. The covered time period differed between lakes and was 200-300 231
years in SS4 and SS1381, and ~30 years in SS1590. All Daphnia clones were putatively 232
triploid, based on the criterion of three different alleles at a minimum of one locus in each 233
multi-locus genotype (MLG). None of the MLGs included a locus with more than three 234
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alleles. Of the eight loci used for genotyping, six (Dp162, Dp291, Dp369, Dp401, Dp437, 235
Dp461) had three different alleles in at least one individual, while two (Dp43, Dp173) had a 236
maximum of two alleles. 237
Clonal diversity differed between the three lakes (Table 1): of 28 eggs examined in SS4, we 238
found a total of three MLGs (maximum of 2 per time period, Fig. 3A) of which one was 239
dominant throughout three centuries (MLG1). Only a single clone (MLG10) was detected in 240
the 21 isolates from SS1381, which persisted over several centuries, as did MLG1 in SS4. In 241
contrast, the Daphnia population in SS1590 was more diverse than the former two lakes with 242
seven MLGs in a total of 10 isolates, of which only one MLG was found during two (non-243
adjacent) time periods. These differences in clonal diversity and dominance structure are also 244
reflected in the rarefied estimate of clonal diversity (eMLG, Table 1), which in SS1590 was 245
about 3.5 and 6 times higher than in SS4 and SS1381, respectively. No clear temporal pattern 246
was discernible in either of the two lakes with long-term records (Fig. 3A, SS4 and SS1381). 247
The Daphnia lineages of the three lake populations formed well-defined clusters (DAPC, Fig. 248
3B), separating the lakes at almost equal distances. The analysis also illustrates the genetic 249
similarity of three MLGs of SS4. While the clones of SS1590 were more scattered they still 250
formed their own cluster without overlap to the other lakes. 251
252
Discussion 253
The results of this study indicated almost equal separation of the three Daphnia populations 254
among the lakes (Fig. 3) and despite considerable in-lake and regional environmental change 255
since ~1700 AD (discussed below), the clonal structure of Daphnia populations in two of 256
three lakes (SS4 and SS1381) was remarkably stable. Importantly, we found strong 257
dominance of a single, lake-specific asexual clone throughout the past two to three centuries 258
in each of the two lakes. 259
260
Spatial patterns 261
The analysis of population genetic structure revealed almost equal separation of the three 262
populations (Fig. 3), a spatial pattern that may reflect the primary environmental differences 263
among the lakes as well as the historic connectivity between these lakes: SS4 is oligosaline 264
and thus differs strongly from SS1381 and SS1590. The latter two are quite similar to each 265
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other in terms of their water chemistry (Table S1) and typical of many of the freshwater lakes 266
around the head of the fjord [33]. Clearly, ionic composition and inorganic C chemistry is an 267
important driver of genotypic variability as much as it determines regional biodiversity and 268
community structure (diatoms, chrysophytes; [51,52]. Conductivity is the dominant control on 269
algal composition in this area and is highly correlated with both DOC concentration and 270
CDOM quality [37]. Conductivity was also identified as one of the major factors driving 271
clonal composition of Daphnia populations in an area about 250 km north of Kangerlussuaq 272
[30]. Lakes SS1381 and SS1590 have never been connected hydrologically, since SS1381 lies 273
outside the boundary of the large palaeolake that included SS4 in the period immediately after 274
deglaciation [41]. The lack of shared clones between lakes suggests local adaptation to the 275
environmental conditions of each resident lake, in particular given the geographic proximity 276
between all three study lakes (less than 10 km) and a likely lack of dispersal limitation as 277
observed in other populations of West Greenland Daphnia [29]. 278
The overall greater clonal diversity of the SS1590 Daphnia population compared with that of 279
SS4 and SS1381 may reflect the greater diversity of habitats in this lake – its two sub-basins 280
are quite different in terms of their morphometry and substrate variability, including the 281
dominant macrophytes (Anderson, unpublished field observations). Not only are littoral and 282
benthic habitats more diverse in SS1590, but the lake also has a more dynamic response to 283
evaporative driven lake level changes than many of the lakes in the area. These short-term 284
lake level changes also exacerbate habitat heterogeneity more at this site than others, due to 285
its morphometry. SS1381 has a simpler morphometry, essentially with one deep basin. SS4, 286
the largest of the lakes can be considered a pelagic system and while lake levels are variable 287
here as well, the contribution of the littoral zone to whole lake production and diversity is 288
probably more limited. 289
290
Regional environmental change 291
Lakes in the Kangerlussuaq area are tightly coupled to regional climate change which 292
influences both terrestrial landscape and in-lake aquatic processes, such as hydrological 293
runoff, terrestrial productivity, lake levels, conductivity and DOC concentration [53]. The 294
period covered by the genetic analyses, ~300 years, includes the end of the Little Ice Age 295
(LIA); a period of considerable change in regional climate, with aridity and fluctuating 296
temperatures. Lake levels would have been lower than present [41,43] and aridity affects dust 297
deflation from the sandurs immediately north and to the east of the study area. As the lakes 298
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are strongly P-limited [54], dust is a possible important nutrient source. Atmospheric reactive 299
N deposition has increased across the Arctic [55] and Greenland is no exception. The 300
changing nutrient balance associated with these varying sources will have impacted primary 301
production and thus secondary producers (i.e. Daphnia). But as well as these broader, 302
regional responses which can be traced across lakes, each individual lake has its own 303
ecological trajectory in environmental space (see for example [35]). Long-term air 304
temperature records show considerable variability during the 20th century, including a period 305
of pronounced cooling. Rapid warming characterizes the start of the 21st century. Lake 306
temperatures track air temperatures tightly in this area [56] and so epilimnetic temperatures 307
would have varied similarly. 308
As an example for local environmental variability since LIA, SS4 showed pronounced 309
variation of okenone, a pigment which can be used as a proxy for purple sulphur bacteria 310
(PSB) abundance [34]. In SS4, in conjunction with indicators of primary production, variation 311
of ephippial densities provides evidence of historic changes in trophic interactions. Ephippial 312
production serves as a rough estimate of Daphnia population size [57,58]. There is some 313
suggestion that over the last ~800 years the Daphnia population in SS4 has been tracking 314
purple sulphur bacteria (Frisch & Osburn unpublished), which Daphnia may exploit as a 315
direct or indirect food source, as observed in other studies [59,60]. 316
317
Long-term persistence of dominant clones 318
Despite these profound environmental changes, there is limited genotypic variability over 319
time. We did not find evidence for environmental dynamics to drive patterns of population 320
genetic structure or genetic diversity over the past several centuries. Our data suggest the 321
presence of a single clone in two of the study lakes, in SS4 (MLG1) and SS1381 (MLG10), 322
across all sample depths, over the past 200 to 300 years. In SS4, two other clones were 323
detected in two different time periods, both with very low abundance. In contrast, the third 324
lake (SS1590) had a higher number of clones. While it is obvious that clonal diversity in this 325
lake overall was much higher, the temporal pattern of genetic structure and diversity cannot 326
be identified with certainty due to the low numbers of isolates; overall ephippial density in 327
this lake was much lower (personal observation) with a smaller amount of well-preserved 328
eggs suitable for genetic analyses. 329
330
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There are several tenable explanations for the apparent dominance and persistence of single 331
clones: 332
(1) The persistent clone may be a general-purpose genotype that successfully exploits a 333
variety of historic environments that are not (yet) within a harmful range 334
The invasion and dominance of a single, asexual Daphnia clone has been observed in several 335
lakes throughout a wide geographic range. For example, an asexual Daphnia pulex clone 336
invaded the population in Lake Naivasha, Kenia in the 1920s, and then displaced the local 337
sexual, genetically diverse Daphnia population [20]. Successful invasion of the same clone 338
was observed throughout Africa in a wide range of aquatic habitats and environmental 339
conditions, testifying to the exceptional niche breadth of this asexual clone. Similar 340
observations have been made in Japan [61], where four asexual Daphnia clones invaded a 341
large number of aquatic habitats across a wide geographical range and ecological conditions. 342
In our study of Arctic populations, the persistence of Daphnia clones despite major 343
temperature changes over the last centuries in the study area may reflect the considerable in-344
lake temperature gradient that Daphnia experience on a regular basis. At SS4, daily vertical 345
migration to graze directly or indirectly on PSB and POC (DOC) in the metalimnion at SS4 (a 346
distance of some 8-12 m) would expose animals to a temperature change of >10 °C, 347
considerably more than the temperature change during the recent millennia. Moreover, the 348
annual temperature range in the epilimnion is also in the order of 10 °C [44]. Although only 349
SS4 has a metalimnetic PSB plate, all three study lakes have substantial gradients in DOC and 350
POC, suggesting that they are utilising a microbial loop in the lakes. The three study lakes 351
are seasonally anoxic with hypolimnetic reductions in O2 during both summer and winter. It is 352
possible that these seasonal environmental changes and the vertical O2 gradients exert greater 353
physiological stress in Daphnia than the stress associated with regional warming. Of course, 354
this genetic stasis may not continue should environmental change in SW Greenland stay at a 355
similar rate [31]. 356
(2) Undetected by the applied genetic resolution, a single MLG may comprise several distinct 357
clonal lineages, each adapted to a specific historic environment 358
The possibility of cryptic genetic variation must be considered due to the limited resolution 359
offered by microsatellite markers which cannot fully account for possibly existing genome-360
wide variation. For example, using 12 microsatellite loci, So et al. [61] detected only four 361
MLGs in asexual Daphnia pulex that had invaded Japanese lakes and ponds. Interestingly, 362
these MLGs comprised 21 mitochondrial haplotypes, indicating a higher genotype diversity 363
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than that resolved by the microsatellite loci [61], a finding that was later confirmed using 364
whole genome sequencing [62]. In particular, these authors concluded that the observed 365
divergent traits in these closely related asexual genotypes evolved without recombination 366
from an ancestral clone by a limited number of functionally significant mutations [62]. 367
(3) Epigenetic modifications explain clonal adaptation and allow clones to persist 368
There is increasing evidence that epigenetic mechanisms contribute to evolution [63,64] and 369
to adaptive responses related to climate and environmental change [65–67]. In the absence of 370
genomic variation, it is becoming increasingly evident that changes in epigenetic profiles 371
could allow for rapid, heritable adaptations to environmental cues that precede the more 372
slowly evolving changes in DNA sequences [68], i.e. the classical Darwinian mutation 373
accumulation and selection concept. Epigenetic modifications would allow persistence of 374
asexual clones in the absence of recombination and generation of genetic variation, because 375
transgenerational inheritance of DNA methylation is highly likely in Daphnia, whose gametes 376
are derived from almost fully matured tissue [69]. 377
378
Synthesis 379
In order to test the ecological implications of these findings, laboratory experiments are 380
needed in future studies. In particular, representatives of the current populations can be used 381
to test whether the dominant clones exhibit phenotypic plasticity with a wide tolerance 382
towards local conditions (temperature, food, salinity), and whether local adaptation to these 383
conditions can be observed. If possible, hatchlings of eggs from older sediments should be 384
included to compare phenotypic and transcriptomic responses of ancient and contemporary 385
isolates [12,70]. In addition, the role of epigenetic modifications should be considered in 386
further studies. 387
These results, to our best knowledge, are the first to report the population genetic structure of 388
asexual, polyploid Daphnia populations of the circumpolar Daphnia pulex-complex over 389
century timescales. Future, more intensive sampling may reveal additional clonal lineages; 390
however, the observed dominance patterns and persistence of individual clonal lineages are 391
unlikely to change. Our results stimulate several questions relating to the mechanisms of 392
adaptation in these populations, as well as their evolutionary fate during the next decades and 393
beyond, on the assumption that climate change in Greenland and other Arctic regions 394
proceeds on the predicted trajectory with severe ecological consequences. 395
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 24, 2020. ; https://doi.org/10.1101/2020.07.22.208553doi: bioRxiv preprint
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396
Acknowledgements 397
We are grateful to Erika Whiteford, Madeleine Giles, Amanda Burson and Tania Cresswell‐398
Maynard for help with field work, to Chris O'Grady for assistance in the laboratory, to Andy 399
Moss for providing laboratory space for core processing at UoB and to Fengjuan Xiao 400
(Lboro) for the loss-on-ignition analyses. DF received funding from the European Union’s 401
Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant 402
agreement No. 658714. NJA’s work in Kangerlussuaq is the supported by NERC. 403
404
Supplementary Information 405
Fig. S1 Photograph of a sediment core recovered in SS4 406
Fig. S2. A-B. Correlation of replicate cores at SS4 C. Core correlation at SS1381 407
Table S1 Nutrient and major ion water chemistry in the three study lakes 408
Table S2 Details on allelic diversity for eight microsatellite loci of the three Daphnia pulex 409
s.l. populations in this study 410
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Table 1. Genetic diversity of the Daphnia pulex population in three lakes of West Greenland 411
integrated over several sediment depths. 412
413
414
415 Pop N MLG eMLG SE
SS4 28 3 1.7 0.7 SS1590 10 6 6.0 0.0
SS1381 21 1 1.0 0.0 Total 59 10 3.9 1.1
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Fig. 1 Overview of the study area. Insert shows location of the study lakes SS4 (Braya Sø), 416
SS1590, and SS1381. 417
418
419
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Fig. 2 Environmental history at SS4 (Braya Sø) and the response of Daphnia genotypes. A. 420
Climate variability: reconstructed summer lake water temperatures based on alkenones at 421
Braya Sø and Lake E (see [44] for details) and the mean annual air temperature recorded at 422
Nuuk. B. Aquatic production indices for the lake: organic C burial reflects total in-lake 423
production and abundance of purple sulphur bacteria inferred from porewater fluorescence (as 424
Parafac Component C2). C. The stability of the dominant genotype over time period covered 425
by the sediment analyses is indicated by the solid red line; two secondary, minor genotypes 426
(GT) are indicated (solid orange and dotted red lines). 427
428
429
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Fig. 3 Spatial and temporal population genetic structure of the Daphnia populations in three 430
lakes. To avoid an artificial increase of identical genotypes, only one egg per ephippium was 431
used for microsatellite analysis. A. Abundance of multilocus genotypes (MLG1 to MLG10) 432
with information on lake and sediment age from which eggs were isolated. Each row 433
represents a lake: SS4 (4 time periods), SS1381 (4 time periods), and SS1590 (three time 434
periods). B. DAPC of MLGs identified in the three lakes and time periods. Fill colours 435
represent MLGs and correspond to colours in A. 436
437
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(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 24, 2020. ; https://doi.org/10.1101/2020.07.22.208553doi: bioRxiv preprint
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Competing interests statement 647
The authors declare no competing interests. 648
649
Authors’ contributions statement 650
DF conceived the study. MD conducted the molecular work and analysed the molecular data 651
with DF and input from JKC. NJA and CLO analysed the environmental data. DF and NJA 652
conducted the field work, interpreted the data and wrote the manuscript with input from all 653
authors. All authors gave final approval for publication and agree to be held accountable for 654
the work performed therein. 655
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 24, 2020. ; https://doi.org/10.1101/2020.07.22.208553doi: bioRxiv preprint