4.1. Biogeochemical Processes Involved in Varve Formation: Where, When, and How?
Processes driving varve formation are remarkably variable and site-specific. Consequently, detailed knowledge of the local processes that promote and modulate particle flux dynamics to the sediment for each site becomes essential. This knowledge is a pre-requisite for proper paleoenvironmental and paleoclimatic interpretations of the sedimentary archive [
17,
38] and for building reliable varve-based chronologies. An independent absolute varve chronology was obtained from the composite sedimentary sequence of both sediment cores extending from 2012 back to Common Era (CE) 1347 [
27]. The varves of Lake Montcortès are formed by couplets of white calcite and brownish organic layers. The white layer was assumed to have deposited during spring/summer and the dark layer in fall/winter [
17,
27]. Biogenic varve formation is highly complex as it adds physical and chemical processes to the complex biological processes involved [
15].
With this modern analogue study several goals are pursued for Lake Montcortès: (i) to identify the main limnological and sedimentological mechanisms leading to varves formation; (ii) to establish in which season(s) the deposition of individual calcite sub-laminae occurs and (iii) to determine if the obtained results are coherent with the varve structure and previously proposed mechanisms of their formation [
26].
Elevated Ω values were recorded in the epi- and metalimnion Lake Montcortès from late spring to early fall, suggesting that calcite precipitation mostly occurred during this period. The deposition rates were highest in summer when photosynthetic organisms were most active (
Figure 2A,B). Additionally, higher POC values were observed during this period, indicating that the main origin of calcite crystals were newly formed autochthonous CaCO
3 precipitates (
Figure 2C).
In fact, the presence of blocky, polyhedral calcite crystals was confirmed in all sediment trap samples and showed a clear seasonal pattern, being smaller when formed in spring and summer (
Figure 2D). Deposition of smaller crystals was not only concurrent with Ω, but also with larger fluxes of very tiny diatoms (~10 µm), suggesting that phytoplankton cells could be acting as calcite condensation nuclei under enhanced calcite Ω, pH and productivity conditions. Other sources of calcite input, such as detrital carbonates from the catchment, were unimportant for the studied period.
Other seasonal events are precipitation of calcite and OM (
Figure 2C). Both variables displayed maximum fluxes in summer to fall. However, calcite precipitation shows the lowest values from winter to spring and was exceeded by OM deposition suggesting a match between the timing of contemporary precipitation of calcite and OM deposition and the alternating couplets of white calcite and brownish organic sublayers of the sediment which would correspond to summer/fall and winter/spring, respectively. These results did not completely back previous paleolimnological inferences that assumed that the light calcite layer was formed in spring/summer and the organic layer was formed in fall/winter [
26]. Because of the lack of local or regional modern analogue studies, the authors relied on available literature [
17] to explain the observed mechanisms of varve formation.
According to our results, the process of varve forming during the period of study would probably match one of the varve patterns described in the sediment record of Lake Montcortès, once deposited although this remains to be further confirmed. The seasonal calcite-crystal distribution observed in our quarterly sediment traps would produce calcite sub-layering with coarsening upward, i.e., fine-grained calcite crystals deposited in summer and coarse-grained calcite crystals in fall. This texture is often observed in the sedimentary sequence from CE 1350 to 1850 [
26]. Modern analogue studies of crystal sizes in calcite sublayers may allow inferring environmental conditions at seasonal resolution, which would be of paleoclimatic significance.
4.2. Is Contemporary Seasonal Deposition of Pollen Consistent with a Two-Layer Varve Model?
The formation of annually laminated lake sediments relies on seasonal deposition linked to climate and on the related flux of biotic and abiotic matter of multiple autochthonous and allochthonous sources to the sediment. Thus, pollen from the catchment and beyond arrives and is buried in lacustrine sediments. Pollen data have been widely used to reconstruct past climate change and have provided insights into the influence of intra-annual weather conditions to pollination patterns. We now argue that combining pollen data from sediment traps and varved lake sediments with modern meteorological data may produce long, high-resolution ecological time series. An example of what can be achieved with this approach are the continuous, long-term and high-resolution climate series obtained by associating paleoclimate data derived from tree rings and other similar proxies with instrumental climate measurements, at annual and seasonal resolutions [
39]. To empirically support this hypothesis, this study is primarily intended to identify modern seasonal depositional patterns of pollen in sediment traps deployed in the varved Lake Montcortès that would be useful for interpreting subfossil pollen records from the same lake.
Pollen maxima were recorded during spring, concurrently with the flowering season of the involved taxa, and preceded the temperature and precipitation maxima in summer by one month (
Figure 3). Total pollen influx and taxonomic composition show a strong seasonal signal for the study period, peaking positively during spring/summer and negatively during fall/winter. The major components of the pollen assemblages,
Pinus (pine) and
Quercus (oak), dominate the pollen counts and match seasonal trends in temperature and precipitation, with
Pinus almost tripling
Quercus percentages in spring/summer but falling to similar levels in fall/winter. As far as the remaining species are concerned, most significant differences were observed in
Plantago, Chenopodium, Typha/Sparganium, Cyperaceae,
Fraxinus and
Juniperus/Cupressus, which prevailed in the spring/summer assemblage, and
Cannabis and
Corylus, which were more abundant in the fall/winter assemblages (
Figure 3). Pollen of
Cannabis (hemp) prevailed during the fall.
Cannabis is a cultivated plant with pollen being present and fairly abundant in the surroundings of Montcortès for the last 1200 years. Nonetheless, we still have not been able to locate the exact source of that pollen [
33,
34].
Canonical correspondence analysis (CCA) shows the first two axes accounting for 70.7% of total variance. The strongest gradient coincides with axis 1 (56.8% of the total variance), which was highly and positively correlated with relative humidity and atmospheric pressure and negatively correlated with wind velocity (
Figure 4). Along this gradient, pollen samples became aligned from spring (left) to winter (right) suggesting a seasonal succession with summer and fall occupying intermediate positions. Pollen taxa were ordered according to the same gradient. The spring/summer group was highly correlated with temperature, precipitation and wind velocity. The fall/winter group was also highly correlated with wind direction from WSW.
Specific aspects of pollen sedimentation require further attention. For example, a lag in pollen sedimentation was observed between production and deposition throughout the year. This lag can have several causes; for example, the influence of internal water dynamics and resuspension, or the fact that soils can be washed into the lake along several successive months after pollen deposition. With respect to lake internal processes, it is worth mentioning that strong thermal water column stratification occurred from March to November 2014 coinciding with the main pollen production period. This stable stratification could have slowed pollen sedimentation rates, whereas in winter vertical mixing of the water disrupted the thermal stability in January and February 2015, facilitating a release of pollen to the lake bottom several months after their production.
The similarity of sedimentation patterns between
Pinus and
Quercus pollen was unexpected, because their respective pollen grains show important morphological differences that make their capabilities for air suspension significantly distinct. However, once the pollen is submerged in the lake water of Lake Montcortès, the settling of both types of pollen was quite similar even during summer, when the thermal stratification was stable. This finding suggests that internal lake dynamics neutralise the effect of such differences and that resuspension or catchment runoff may become more important about pollen sedimentation. In general, the seasonal pattern in pollen deposition coincided well with the suggested two-layered varve model. In summary, pollen analysis was able to identify two well differentiated modern assemblages, one corresponding to spring/summer and the other representing fall/winter. This matches well to the seasonal pattern identified in sedimentological (varve) studies [
41]. Thus, utilising appropriate transfer functions supports the hypothesis that the combination of pollen data from sediment traps, varved lake sediments and modern meteorological data may produce long and high-resolution ecological time series.
4.3. Revisiting Meromictic Lake Montcortès: Has the Mixing Regime Changed?
Oxygenation of lake water relies on oxygen diffusion from the atmosphere, addition from autochthonous primary production and external inputs of oxygenated water. Lake mixing is the main mechanism, which distributes oxygen throughout the water column and is therefore central for lake health. Mixing regimes can shift with climate changes [
42] and human activities [
43]. Recently, dissolved oxygen depletion leading to hypoxia or even anoxia has become a matter of concern around the world, and there is a high potential for deterioration under the current global warming [
44]. Furthermore, it is suggested that climate change will favour cyanobacteria, which form unpleasant blooms and produce toxins that are harmful to humans [
45].
At the same time, obtaining time series of instrumental oxygen records that extend beyond a century is problematic, thus, reducing the possibility of recording long-term changes in oxygen variations that could be associated with climate or human influence. One seminal exception is Lake Zürich, where 50 year long-series of temperature and oxygen profiles corroborated that the increase in the thermal stratification period tends to favour reduction in the homeothermal brake and consequently favours the onset of hypoxia or anoxia [
46,
47]. Where concurrent series of oxygen and temperature are not available, the combination of proxies of oxic/anoxic conditions with high-resolution paleolimnological records can result in a useful surrogate to assess long-term changes in oxic/anoxic shifts through time.
The varved sediment of LM appears to be highly suitable to provide such high-resolution data. Since redox processes depend on the biogeochemical conditions of lakes and the chemical characteristics of the involved compounds, we firstly conducted a modern analogue study consisting of a monthly survey of main redox indicators, i.e., DO, Fe, Mn and marker pigments [
48,
49,
50,
51], between October 2013 and October 2016. The same type of marker pigments was also examined in sediments: isorenieratene (Chlorobiaceae) as a tracer of euxinia, okenone (Chromatiaceae) as indicators of anoxia and oscillaxanthin (Oscillatoriales) as a general marker of Cyanobacteria that often points to increased eutrophic conditions. Furthermore, we used selected elemental ratios of the sedimentary record to survey shifts in the oxic/anoxic conditions of the lake over the last 500 years: bromine (Br) as a tracer of organic matter [
52], the Ca/Ti and Si/Ti ratios as indicators of biologically mediated calcite and silica production, respectively, and the S/Ti and S/Fe ratios as indicators of the presence of sulphur compounds [
53].
Lake Montcortès is reported to be meromictic for years [
54]; nonetheless, we observed brief episodes of complete mixing during the winters of 2013 and 2014 that triggered precipitation of Fe and Mn oxides. With the onset of anoxic conditions, precipitated Mn and Fe oxides started to redissolve and sulfur bacteria grew in the hypolimnion under anoxic conditions, when Fe and Mn reached maximum concentrations. The oscillaxanthin was absent in the water column during the sampling period. Such conditions endured over the year 2016, which was a non-mixing year. These results confirmed that redox proxies were working well and provided the information required to properly interpret the sedimentary record. Applying this knowledge to the results obtained from sediment analyses, we were able to distinguish four prevailing situations on the basis of selected redox proxies: (A) years with abrupt and substantial sediment input (turbidites); (B) years with mixing and oxygenation of the water column; (C) years with strong stratification, anoxia, intense activity of sulfur bacteria and increased biomass production; (D) years showing stratification and anoxia, but relatively low biomass production (
Figure 5). Interestingly, approximately 45.3% of the years were monomictic years, which mostly occurred in groups of several consecutive years. Meromictic years show a similar pattern. Most (A) years coincide with the climatic instability of CE 1850–1899. The (B) years were rather scattered but were best represented between CE 1820–1849. Most (D) years happened from CE 1500 to 1820, when human activities were locally most intense for the studied period. Almost all (C) years belong to the 20th century. More than 90% of the years with climatic instrumental records belonged to B and C.
4.4. Are GDGTs Promising Indicators of Seasonal Temperature Shifts?
Branched glycerol dialkyl tetraethers (brGDGTs) are bacteria derived and widespread lipids of terrestrial and aquatic environments. The global distribution of brGDGTs in soils and peats has been associated with past temperature, and they are therefore used as proxies of mean annual temperature (MAT proxies) in such environments [
55]. However, in some regional studies, brGDGTs indices appeared to be influenced by other variables, e.g., precipitation, humidity and soil properties [
56]. This proxy was originally calibrated against annual averages of environmental variables. However, a hypothesis arose that these proxy estimates are biased towards particular seasons, because the bioproduction of brGDGTs is enhanced under the most favourable conditions, which occur mostly in summer.
To answer whether soil brGDGT proxy estimates are seasonally biased, the brGDGT distributions and the brGDGT-derived MAT estimates were examined in settling particulate matter (monthly traps) and surface soil samples from the catchment area of Lake Montcortès (MAT: −3.3 to 17.6 °C). No clear-cut seasonal pattern of the brGDGT distribution has been found in soils (
Figure 6a), probably because of the slow turnover time in terrestrial environments, which is on timescales of decades or longer. These results confirm previous findings from mid-latitude soils showing that brGDGTs’ distribution and some of the brGDGT-derived proxy measurements are relatively stable through the year [
57]. However, we found that annual shifts in abundance of brGDGTs were controlled by the variability of specific regional factors, i.e., soil humidity and their pH [
13].
The results record seasonal trends in sediment flux from the catchment area. In the case of particulate matter, heavy rain is the main factor influencing the brGDGT abundance, resulting in seasonal variations of brGDGT flux (
Figure 3 and
Figure 6b).
In terms of modern analogues, this study offers evidence that the signatures of brGDGTs in depositional environments are representative of soils in the catchment, and that the accuracy of the derived temperature estimates based on methylation/cyclisation (MBT/CBT) proxies in soils depends on soil properties, that in turn depend on regional factors. Additionally, those brGDGT signatures of soils in the catchment probably represent average environmental conditions over decades or longer, and this observation means that any derived proxy reconstructions can be used only to infer variability in environmental variables over the same timescales. The absence of seasonality in brGDGT proxies from the sediment traps Lake Montcortès is due to the lack of significant “in situ” lacustrine production of brGDGTs (
Figure 6c). The most straightforward explanation is that the main sources of brGDGTs are catchment soils, and that their non-seasonal signals are transferred to the settling particles, which have a distribution of brGDGTs that appear to be a weighted mean of soil signals (
Figure 6c). In addition, similar patterns of brGDGT fractional abundance in soils and sediment traps confirm soil-related sources to the particles in sediment traps (
Figure 6d). It remains unclear if downcore variability in brGDGTs at an annual scale will also capture changes derived from in situ production, and if derived brGDGT proxies will be suitable to build high-resolution MAT reconstructions.
4.5. From Contemporary Phytoplankton to Subfossil Pigments, What Can We Learn about Community Changes?
In general, qualitative and quantitative relationships between modern climatic variables, aquatic primary producers and their pigments and subfossil marker pigments of the sediment have received little attention despite their potential to build reliable climatic proxies. To help fill this gap, we performed a modern analogue study to determine how phytoplankton and marker pigment information compare with the sedimentary record. For this purpose, we applied an analogue matching (AM) technique, which is concerned about identifying contemporary sites that most closely match the species assemblage identified in the past [
7].
We assessed and compared the annual cycle of phytoplankton in the epi- and metalimnion and of marker pigments in the epi-, meta- and hypolimnion, and also in a sediment trap deployed at 20 m water depth. Redundancy Analysis (RDA) was applied to identify potential associations between the phytoplankton taxa identified by microscopy. Then, we performed a similarity analysis in order to examine expected bias between modern and subfossil samples (CE 1493–2013) in terms of presence or absence of common pigment markers.
The total biovolume of phytoplankton depicted a regular annual cycle with growing peaks in early spring and summer and low biomass in winter (
Figure 2B). Phytoplankton biovolume of the metalimnion was highest in summer (>6 × 10
6 µm
3/mL). Centric Bacillariophyceae and Chlorophytes follow one another as the dominant taxa in 2013–2014, whereas smaller centric Bacillariophyceae took over during 2014–2015 growing periods (
Figure 7).
The RDA analysis allowed to identify marker pigments more tightly associated with each taxonomic group (
Figure 8). Chlorophyta, Dinophyta, Cyanoprokaryota (Cyanobacteria and Cyanophyta), centric Bacillariophyceae, Volvocales and Cryptophyta biovolumes explain a significant percentage of variance in marker pigment concentration, while the percentage variance explained by the other taxonomic groups was insignificant. Each taxonomic group was associated with specific marker pigments. The biovolume of Chlorophyta was associated with the marker pigments neoxanthin, lutein, chlorophyll-b and zeaxanthin, the biovolume of Dinophyta mainly with peridinin, diadinoxanthin, fucoxanthin, Chl-c1 and c2, although Chl-c2 was also partially associated with the biovolume of Cryptophyta. The biovolume of Cryptophyta was mainly associated with alloxanthin and a-carotene, both partially explained by pennate Bacillariophyceae. We know from the literature that some of the pigments associated with Dinophyta are also present in Bacillariophyceae (e.g., fucoxanthin, diadinoxanthin, Chl-c1 and Chl-c2), which is coherent with the observed RDA biplot association. Cyanobacteria biovolume was associated with myxoxanthophyll-like, while association with marker pigments of phototrophic bacteria is due to co-occurrence in the samples with these groups. These data are essential to identify pigments with a higher potential to be used as modern analogues in the sediment record.
All this information is relevant to better identify pigments from sediment traps and use them as modern analogues for the sediment record following taxonomic affinity criteria and preservation.
The concentration of most representative marker pigments was calculated for the entire water sampling period (2013–2015), in order to compare their spatial variations between epi-, meta- and hypolimnion, as well as with their deposition in the sediment trap (
Figure S1,
Supplementary Materials). Marker pigments of Bacillariophyceae (fucoxanthin:
Figure S1c) and Dinophyta (diadinoxanthin:
Figure S1d) were produced and deposited in higher amounts in spring and summer while Chlorophyta marker pigments (Chl-b, lutein and zeaxanthin:
Figure S1e,f,h) were produced mainly in fall, and marker pigments of Cryptophyta (alloxanthin:
Figure S1g) were produced throughout the year with the exception of fall. Zeaxanthin is a marker pigment also indicative of Cyanobacteria and decoupled from parent marker pigments of Chlorophyta during summer, it indicates a higher contribution of Cyanobacteria during summer months (
Figure S1). Marker pigments of photosynthetic sulphur bacteria were only found in the hypolimnion during summer and fall (
Figure S1i,j).Most marker pigments from the water column were also identified in the sediment record. A list of the marker pigments co-occurring in the water column and sediment record is given in
Table S1 (
Supplementary Materials). Their taxonomic affinities are also shown and inform about their likely biological sources, which are consistent with the taxonomic phytoplankton groups of the epi- and metalimnion (
Figure 7). To gauge the similarity between water column and sediment samples we applied the Sorensen index (S) on a similarity matrix built with binary data. This very simple similarity index provides a greater “weight” to species common to the samples than to those found in only one sample. According to S, epi-, meta- and hypolimnetic samples resemble at ≥80%, whereas meta and hypolimnetic samples resemble each other relatively more (92%), likely because of lower oxidation rates during anoxic conditions of the hypolimnion. Interestingly, when compared with sediment samples of the top of the core (CE 2007–2013 E), S diminished abruptly to 0.5, that is, the bias was 50% (
Figure 9). This decrease was followed by a slower decrease of S, until values of 30% (circa 1850) and its posterior stabilisation at values of 40–60% that lasted circa two centuries. A Venn diagram highlights that past communities only have a partial modern analogue (
Figure 9) [
5]. These results indicate that approximately 50% of the marker pigments in the water column were destroyed between deposition and permanent burying in the first centimetres of the sediment record, whereas the remaining 40–60% were accurately represented over the period of study. This pattern is roughly consistent with the three phases of sediment loss proposed by [
48]: (1) rapid oxidation, enzymatic metabolism and digestion by herbivores through the water column (half-life T1/2 = days) would account for a breakdown of pigments while they sink, (2) slower post-depositional loss in surface sediments would result from structural rearrangements, release of labile compounds and further oxidation (T1/2 = years) and (3) once buried pigment degradation would have continued at a very slow pace (T1/2 = centuries).
Finally,
Table S2 (
Supplementary Materials) shows which marker pigments were found to be exclusive from present-day samples (6) and which were found only in subfossil samples (7). Some of the unspecific chlorophyll derivatives are produced under distinct environmental conditions and by different mechanisms with time, chiefly through losses of Mg
+2, herbivory, viral attack and enzymatic catalysis [
48]. Noteworthy, however, is the presence of four Cyanobacterial markers in the subfossil record that were not present in modern samples, suggesting either a detection limit for the identification of these pigments in the water column or that past lake conditions were favourable to a high diversity of Cyanobacterial groups including N
2-fixing. In fact, massive blooms of filamentous
Planktothrix rubescens De Candolle ex Gomont have been reported to have flourished in Lake Montcortès in the 1970s [
54]. However, based on microscopical counts and marker pigment detection, Cyanobacteria seem to constitute only a low percentage of the phytoplankton currently thriving at Lake Montcortès (
Figure 6), an issue that needs further confirmation.