Long-term dynamics of microbial eukaryotic communities from lakes

Since the industrial revolution, Lake Biwa (Japan) has been subjected to multiple stressors of human origins causing alterations in the composition and function of its resident biota and thus its ecosystem services. Lake eutrophication in the 1960s, the manipulation of lake water level since 1990s and concomitantly rising of water temperatures have been reported as strong environmental drivers of changes for both the piscivorous and planktonic assemblages. However, changes in the huge diversity of microbial taxa that inhabits Lake Biwa have been still understudied. The sequencing of the DNA preserved in sedimentary archives is a promising approach to provide long-term time series of microbial assemblages. Here, we applied a 18S metabarcoding (V7 260 bp) to sedimentary DNA from Lake Biwa. We investigated how the microbial eukaryotic community changed over the last 120 years and which environmental stressors influenced changes in Lake Biwa biota. A total of 2037 microbial eukaryotes OTUs were retrieved including strict heterotrophs (cercozoa, ciliates, fungi, bicosoecida, perkinsea), strict phototrophs (diatoms and chlorophytes) and other groups (dinophyceae, unclassified alveolates, unclassified stramenopiles). Our analysis highlights that the manipulation of the water level and modification in planktivorous assemblages were the major stressors driving changes in the microbial eukaryotic community with a major shift in the 1990s marked by an increase in the relative abundance of diatoms (e.g., Aulacoseira sp) and chlorophytes (Mychonastes sp, Choricystis sp). Overall, our work provides new knowledge about the effects of multiple stressors on lake ecosystems biota.

Historical deposits of sedimentary DNA are a promising target for molecular tools with potential to inform about long-term changes in aquatic microbiome (i.e., bacteria, archaea protists, fungi, viruses) and how microorganisms are controlled by viral infection, pathogens, and larger predators (e.g. zooplankton and fish). As sedimentary DNA archives can encompass timescales that span decades to hundreds of thousands of years, they complement and enhance contemporary data derived from water monitoring. The data from such historical monitoring is integrative and enable ad hoc assessment of biological responses to past environmental and more recent anthropogenic perturbations. Over the last decade, numerous studies based on sedimentary DNA work have used DNA metabarcoding to successfully reconstruct temporal changes in microbial communities, including cyanobacteria and microbial eukaryotes. We will present an overview of the work of the five authors as well as the global scientific community, which will reveal the potential to answer unique research questions that can only be provided when including ancient sedimentary DNA for the reconstruction of long-term temporal changes in aquatic microbial communities. We will then discuss the limitations of sedimentary ancient DNA to reconstruct past microbial communities, provide perspectives on future challenges for the field, and introduce novel approaches for studying past microbial ecosystems in sedimentary records. For instance, shotgun sequencing allows to distinguish between recent and past microbes in sediments via the presence of post-mortem DNA modifications. In complement, hybridization capture methods lower greatly the detection limit of the DNA derived from taxa of interest and open up new avenues for sedimentary DNA research.

On the annual and interannual scales, lake microbial communities are known to be heavily influenced by environmental conditions both in the lake and in its terrestrial surroundings. However , the influence of landscape setting and environmental change on shaping these communities over a longer (millennial) timescale is rarely studied. Here, we applied an 18S metabarcoding approach to DNA preserved in Holocene sediment records from two pairs of co-located Swedish mountain lakes. Our data revealed that the microbial eukaryotic communities were strongly influenced by catchment characteristics rather than location. More precisely, the microbial communities from the two bedrock lakes were largely dominated by unclassified Alveolata, while the peatland lakes showed a more diverse microbial community, with Ciliophora, Chlorophyta and Chytrids among the more predominant groups. Furthermore, for the two bedrock-dominated lakes-where the oldest DNA samples are dated to only a few hundred years after the lake formation-certain Alveolata, Chlorophytes, Stramenopiles and Rhizaria taxa were found prevalent throughout all the sediment profiles. Our work highlights the importance of species sorting due to landscape setting and the persistence of microbial eukaryotic diversity over millennial timescales in shaping modern lake microbial communities.

The use of lake sedimentary DNA to track the long-term changes in both terrestrial and aquatic biota is a rapidly advancing field in paleoecological research. Although largely applied nowadays, knowledge gaps remain in this field and there is therefore still research to be conducted to ensure the reliability of the sedimentary DNA signal. Building on the most recent literature and seven original case studies, we synthesize the state-of-the-art analytical procedures for effective sampling, extraction, amplification, quantification and/or generation of DNA inventories from sedimentary ancient DNA (sedaDNA) via high-throughput sequencing technologies. We provide recommendations based on current knowledge and best practises.

Long-term time series have provided evidence that anthropogenic pressures can threaten lakes. Yet it remains unclear how and the extent to which lake biodiversity has changed during the Anthropocene, in particular for microbes. Here, we used DNA preserved in sediments to compare modern micro-eukaryotic communities with those from the end of the 19th century, i.e., before acceleration of the human imprint on ecosystems. Our results obtained for 48 lakes indicate drastic changes in the composition of microbial communities, coupled with a homogenization of their diversity between lakes. Remote high elevation lakes were globally less impacted than lowland lakes affected by local human activity. All functional groups (micro-algae, parasites, saprotrophs and consumers) underwent significant changes in diversity. However, we show that the effects of anthropogenic changes have benefited in particular phototrophic and mixotrophic species, which is consistent with the hypothesis of a global increase of primary productivity in lakes.

The temporal trajectory of lake microbial communities is still rarely investigated over timescales that encompass the full history of an aquatic ecosystem and, therefore, its response to global or local long-term environmental changes. Thanks to the development of molecularbased procedures in paleoecology, it is today possible to assess changes in lake microbial communities via the extraction and sequencing of DNA molecules preserved in sedimentary archives. Here we applied an 18S-metabarcoding approach on sedimentary DNA obtained from four Swedish mountain lakes to assess the temporal variations of microbial eukaryotic communities over the last 10,000 years. The coupling of DNA data with sediment geochemistry and paleoclimatic data allow us to pinpoint when and to what extent environmental changes impacted these lake ecosystems. This study highlights the strong impact of catchment geology (bedrock and vegetation cover) on the shaping of lake microbial communities suggesting that - at least in a long-term perspective - species sorting may act more intensely than dispersal in these mountain lakes. While providing novel indications about the pioneer communities of those natural systems, data was also used to evaluate the effects of long-term climatic fluctuations on lake microbial communities, a continuous pressure known to impact such communities but still rarely studied over long-term time scales.

To assess the sensitivity of lakes to anthropogenically-driven environmental changes (e.g., nutrient supply, climate change), it is necessary to first isolate the effects of between-year variability in weather conditions. This variability can strongly impact a lake’s biological community especially in boreal and arctic areas where snow phenology play an important role in controlling the input of terrestrial matter to the lake. Identifying the importance of this inherent variability is difficult without time series that span at least several decades. Here, we applied a molecular approach (metabarcoding on eukaryotic 18S rRNA genes and qPCR on cyanobacterial 16S rRNA genes) to sedimentary DNA (sed-DNA) to unravel the annual variability of microbial community in 40 years’ sediment record from the boreal lake Nylandssjön which preserve annually-laminated sediments. Our comparison between seasonal meteorological data, sediment inorganic geochemistry (X-ray fluorescence analyses) and organic biomarkers (pyrolysis-gas chromatography/mass spectrometry analyses), demonstrated that inter-annual variability strongly influence the sediment composition in Nylandssjön. Spring temperature, snow and ice phenology (e.g. the percentage of snow loss in spring, the timing of lake ice-off) were identified as important drivers for the inputs of terrestrial material to the lake, and were therefore also important for shaping the aquatic biological community. Main changes were detected in the late-80s/mid-90s and mid-2000s associated with increases in algal productivity (see from organic biomarkers), in total richness of the protistan community and in relative abundances of Chlorophyta, Dinophyceae as well as Cyanobacteria abundance. These changes could be linked to a decline in terrestrial inputs to the lake during the snow melt and run-off period, which in turn was driven by warmer winter temperatures. Even if our data shows that meteorological factors do affect the sediment composition and microbial communities, they only explain part of the variability. This is most likely a consequence of the high inter-annual variability in abiotic and biotic parameters highlighting the difficulty to draw firm conclusions concerning drivers of biological changes at an annual or sub-annual resolution even with the 40-year varved sediment record from Nylandssjön. Hence, it is necessary to have an even longer time perspective in order to reveal the full implications of climate change. ivarved that resulted in a less intense spring run-off during the snow melt period. The mid-2000s were also marked by the increase of photosynthetic groups such as Scenedesmaceae, Dinophyceae and Cyanobacteria. Our results show that the inter-annual variability in weather conditions makes the 40-year sediment record from Nylandssjön too short to assess the potential effects of climatic pressures. This highlights the limit of drawing firm conclusions concerning drivers of environmental and biological changes at an annual or even sub-annual resolution, and the need of a longer time perspective to reveal the effects of climate change.

Climate change is a key driver of changes in lakes, especially in northern ecosystems. The structure, composition and metabolism of aquatic communities may be highly sensitive to climate-driven weather variability with possible negative effects on lake functioning and ecosystem services. Ice-covered lakes are particularly interesting because of the substantial modifications in snow/ice phenology related to climate-driven processes exposing lake biological communities to several forcing factors including modifications in terrestrial inputs of organic/mineral matter into lakes. In this study we coupled long-term monitoring data (e.g. temperature, spring snow loss, and lake ice-off timing) with paleo-proxies from an annually laminated sediment record assessed by cutting-edge methods in paleolimnology (pyrolysis GC-MS, high-throughput sequencing of sedimentary DNA). Together with data on sediment composition (geochemistry, varve properties), we investigated the consequences of annual weather variability on terrestrial inputs to the lake and the subsequent responses of aquatic biological communities (here phytoplankton and heterotrophic protists). Our data indicate temporal relationships between temperature and snow/ice phenology, with lower temperatures leading to higher spring snow loss. Then, temporal changes in spring snow loss were related to modifications in terrestrial inputs to the lake, with higher spring snow loss inducing higher terrestrial inputs. Biological communities appeared to be highly correlated to modifications in spring snow loss and terrestrial inputs more particularly during two periods (late-80s/early-90s and mid-2000s) marked by changes in richness and structure of the protistan community and cyanobacteria’s relative abundance. Phototroph groups (i.e. Chlorophytes, Dinophyceae, Cyanobacteria) appeared to be particularly impacted by climate- driven environmental changes. This outcomes of this study are in line with the hypothesis that climate-driven modifications of terrestrial inputs into lakes may be a major forcing factor for biological communities from ice-covered lakes.

Studies based on the coupling of a paleolimnological approach and molecular tools (e.g., sequencing of sedimentary DNA) present a promising opportunity to obtain long-term data on past lacustrine biodiversity. However, certain validations are still required, such as the evaluation of DNA preservation in sediments for various planktonic taxa that do not leave any morphological diagnostic features. In this study, we focused on the diversity of planktonic unicellular eukaryotes and verified the presence of their DNA in sediment archives. We compared the molecular inventories (high-throughput sequencing of 18S ribosomal DNA) obtained from monitoring the water column with those obtained for DNA archived in the first 30 cm of sediment. Seventy-one percent of taxonomic units found in the water samples were detected in sediment samples, including pigmented taxa, such as Chlorophyta, Dinophyceae, and Chrysophyceae, phagotrophic taxa, such as Ciliophora, parasitic taxa, such as Apicomplexa and Chytridiomycota, and saprotrophs, such as Cryptomycota. Parallel analysis of 18S ribosomal RNA (rRNA) transcripts revealed the presence of living eukaryotic taxa only in the top 2 cm of sediment; although some limits exist in using RNA/DNA ratio as indicator of microbial activity, these results suggested that the sedimentary DNA mostly represented DNA from past and inactive communities. Only the diversity of a few groups, such as Cryptophyta and Haptophyta, seemed to be poorly preserved in sediments. Our overall results showed that the application of sequencing techniques to sedimentary DNA could be used to reconstruct past diversity for numerous planktonic eukaryotic groups.

L’eutrophisation et le réchauffement climatique sont reconnus comme des forçages majeurs du fonctionnement des lacs. Toutefois les connaissances concernant la réponse des communautés microbiennes eucaryotes à ces forçages sont encore très lacunaires, alors même que les microbes eucaryotes, porteurs d’une vaste diversité taxonomique et fonctionnelle, sont des acteurs clés des réseaux trophiques lacustres. La pertinence des approches paléolimnologiques pour comprendre les impacts de ces forçages sur les communautés lacustres n’est plus à démontrer, mais aujourd’hui l’intégration des outils moléculaires pour analyser l’ADN archivé dans les sédiments offre des opportunités nouvelles pour reconstituer la dynamique passée de la biodiversité lacustre. Dans ce cadre, en s’appuyant sur le couplage entre paléolimnologie et outils de séquençage massif appliqués à l’ADN sédimentaire, ces travaux ont pour but (i) d’apporter des connaissances concernant la préservation de l’ADN des microbes eucaryotes dans les sédiments lacustres (ii) d’appliquer l’approche de paléogénétique sur des carottes sédimentaires issues de 3 lacs pour révéler la dynamique à long terme (de la décennie au millénaire) des microbes eucaryotes en lien avec l’évolution des conditions climatiques et anthropiques. Les résultats acquis sur le lac du Bourget ont permis de mettre en évidence l’efficacité d’archivage de l’ADN planctonique dans les sédiments récents pour la plupart des groupes eucaryotes (notamment chrysophycées, chytrides, chlorophytes, cercozoaires, ciliés, dinophycées). A partir d’une collection de carottes (issues du lac suédois Nylandssjön), l’effet de la diagénèse s’opérant au cours des premières années d’enfouissement a été évalué, permettant de démontrer que si la richesse taxonomique n’est pas impactée, des variations peuvent être détectées dans la structure de la communauté au cours des 10 premières années d’archivage avec une stabilisation du signal au-delà de cette période. L’approche paléogénétique a, en parallèle, été déployée d’une part à l’échelle du siècle sur deux lacs de même typologie mais ayant subi des niveaux d’eutrophisation contrastés, et d’autre part à une échelle temporelle plus longue (2200 ans) pour deux lacs de typologie contrastée (lac du Bourget, France et Igaliku, Groenland). Les résultats acquis démontrent que des réarrangements des communautés s’opèrent de manière concomitante aux périodes climatiques (réchauffement médiéval, petit âge glaciaire, réchauffement récent), et que le réchauffement climatique au cours des 30 dernières années a plus particulièrement favorisé certains groupes, notamment la richesse et l’abondance des dinophycées (en condition non eutrophe ; lac d’Annecy et du Bourget). Toutefois l’effet de l’eutrophisation est identifié comme le facteur le plus structurant, notamment dans le lac du Bourget (cas d’eutrophisation marquée, ~120 µgP.L-1). La forte influence du niveau d’eutrophisation est détectée sur la communauté eucaryote totale et plus particulièrement sur des groupes spécifiques tels que les chlorophytes et les ciliés. Les réarrangements majeurs de la communauté sont par ailleurs marqués par la mobilisation de taxons rares dans l’assemblage microbien eucaryote suggérant le rôle de la biosphère rare dans la capacité tampon des écosystèmes. Ces travaux pluridisciplinaires comptent parmi les premières études paléogénétiques appliquées aux microbes eucaryotes lacustres, contribuant de manière inédite aux connaissances de leur dynamique temporelle à long terme. Ces études tendent à confirmer le potentiel de ces approches pour reconstituer une vaste diversité de communautés lacustres. Les perspectives qui se dessinent dans la continuité de ces travaux concernent à la fois des aspects méthodologiques autour de la calibration du signal ADN archivé et la nécessité de déployer cette approche pour des lacs (sélectionnés) de typologies et histoires écologiques variées.

Assessing the extent to which changes in lacustrine biodiversity are affected by anthropogenic or climatic forces requires extensive paleolimnological data. We used high-throughput sequencing to generate time-series data encompassing over 2200 years of microbial eukaryotes (protists and Fungi) diversity changes from the sedimentary DNA record of 2 lakes (Lake Bourget in French Alps and Lake Igaliku in Greenland). From 176 samples, we sequenced a large diversity of microbial eukaryotes, with a total 16 386 Operational Taxonomic Units (OTUs) distributed within 50 phylogenetic groups. Thus, microbial groups, such as Chlorophyta, Dinophyceae, Haptophyceae and Ciliophora that were not previously considered in lacustrine sediment record analyses appeared to be potential biological markers of trophic status changes. Our data suggest that shifts in relative abundance of extant species, including shifts between rare and abundant taxa, drive ecosystem responses to local and global environmental changes. Community structure shift events were concommitant with major climate variations (more particularly in Lake Igaliku). However, this study shows that the impacts of climatic fluctuations may be overpassed by the high-magnitude eutrophication impacts, as observed in the eutrophicated Lake Bourget. Overall, our data show that DNA preserved in sediment constitutes a precious archive of information on past biodiversity changes. This article is protected by copyright. All rights reserved.

Paleogenetics provides a powerful framework to reconstruct the long-term temporal dynamics of various biological groups from aquatic sediments. However, validations are still required to ensure the authenticity of the molecular signal obtained from sedimentary DNA. Here, we investigated the effects of early diagenesis on the DNA signal from micro-eukaryotes preserved in sediments by comparing metabarcoding inventories obtained for two sediment cores sampled in 2007 and 2013 respectively. High-throughput sequencing (Illumina MiSeq) of sedimentary DNA was utilized to reconstruct the composition of microbial eukaryotic communities by targeting the V7 region of the 18S rDNA gene. No significant difference was detected between the molecular inventories obtained for the two cores both for total richness and diversity indices. Moreover, community structures obtained for the two cores were congruent as revealed by procrustean analysis. Though most of the eukaryotic groups showed no significant difference in terms of richness and relative proportion according to the core, the group of fungi was found to differ both in terms of richness and relative proportion (possibly due to their spatial heterogeneity and potential activity in sediments). Considering the OTUs level (i.e. Operational Taxonomic Units as a proxy of ecological species), our results showed that, for the older analyzed strata (age: 15–40 years), the composition and structure of communities were very similar for the two cores (except for fungi) and the DNA signal was considered stable. However, for the uppermost strata (age < 15 years), changes of moderate magnitude were detected in the relative abundance of few OTUs. Overall, this study points out that, in Nylandssjön sediments, early diagenesis did not induce marked modifications in the micro-eukaryotic DNA signal, thus opening new perspectives based on the analysis of eukaryotic sedimentary DNA to address scientific issues both in the domains of paleolimnology and microbial ecology. Because this study site is ideal for DNA preservation in sediment (quick sedimentation processes, no sediment resuspension, anoxic conditions at sediment–water interface), the generalization of our conclusions, in particular for less favorable sites, must be considered cautiously.

High-throughput sequencing of sedimentary DNA (sed-DNA) was utilized to reconstruct the temporal dynamics of microbial eukaryotic communities (MECs) at a centennial scale in two re-oligotrophicated lakes that were exposed to different levels of phosphorus enrichment. The temporal changes within the MECs were expressed in terms of richness, composition and community structure to investigate their relationships with two key forcing factors (i.e., nutrient enrichment and climate warming). Various groups, including Apicomplexa, Cercozoa, Chrysophyceae, Ciliophora, Chlorophyceae and Dinophyceae, responded to phosphorus enrichment levels with either positive or negative impacts on their richness and relative abundance. For both lakes, statistical modelling demonstrated that phosphorus concentration ([P]) was a dominant contributor to MECs modifications before the 1980s; after the mid-80s, the contribution of air temperature changes increased and potentially surpassed the contribution of [P]. Co-occurrence network analysis revealed that some clusters of taxa (i.e., modules) composed mainly of Dinophyceae and unclassified Alveolata were strongly correlated to air temperature in both lakes. Overall, our data showed that sed-DNA constitutes a precious archive of information on past biodiversity changes, allowing the study of the dynamics of numerous eukaryotic groups that were not traditionally considered in paleo-reconstructions. This article is protected by copyright. All rights reserved.

The emergence of DNA analyses of lake sediments has opened up many new areas of inquiry, including the study of taxa that were traditionally not considered in paleolimnology because they do not leave distinct morphological fossils. Here, we discuss the potential and challenges associated with the study of DNA in paleolimnology to address critical research questions in lacustrine ecology. We examine some recent applications by highlighting studies that have quantified centennial to millennial-scale dynamics, and that considered a diversity of planktonic groups, including bacteria, phytoplankton and zooplankton. We also summarize the main methodological precautions to be taken into account for implementing these types of DNA analyses. Based on our review of the literature focused on the analysis of DNA preserved in lake sediments, the emerging topics we have identified include: (1) the spread, establishment and effect of invasive species, (2) past fish population dynamics, (3) interactions within lacustrine communities, identified through network analyses, (4) potential application of metabarcoding for transfer functions. There are many new and exciting questions that could be addressed using DNA preserved in lake sediment and this will no doubt be an area of continued expansion in the field of paleolimnology for many years to come.