Sedimentary ancient DNA reveals the impact of anthropogenic land use disturbance and ecological shifts on fish community structure in small lowland lake.

Freshwater fish biodiversity and abundance are decreasing globally. The drivers of decline are primarily anthropogenic; however, the causative links between disturbances and fish community change are complex and challenging to investigate. We used a suite of sedimentary DNA methods (droplet digital PCR and metabarcoding) and traditional paleolimnological approaches, including pollen and trace metal analysis, ITRAX X-ray fluorescence and hyperspectral core scanning to explore changes in fish abundance and drivers over 1390 years in a small lake. This period captured a disturbance trajectory from pre-human settlement through subsistence living to intensive agriculture. Generalized additive mixed models explored the relationships between catchment inputs, internal drivers, and fish community structure. Fish community composition distinctly shifted around 1350 CE, with the decline of a sensitive Galaxias species concomitant with early land use changes. Total fish abundance significantly declined around 1950 CE related to increases in ruminant bacterial DNA (a proxy for ruminant abundance) and cadmium flux (a proxy for phosphate fertilizers), implicating land use intensification as a key driver. Concurrent shifts in phytoplankton and zooplankton suggested that fish communities were likely impacted by food web dynamics. This study highlights the potential of sedDNA to elucidate the long-term disturbance impacts on biological communities in lakes.

A paleoecological investigation of recent cyanobacterial blooms and their drivers in two contrasting lakes.

Cyanobacterial blooms are one of the most significant threats to global water security and freshwater biodiversity. Interactions among multiple stressors, including habitat degradation, species invasions, increased nutrient runoff, and climate change, are key drivers. However, assessing the role of anthropogenic activity on the onset of cyanobacterial blooms and exploring response variation amongst lakes of varying size and depth is usually limited by lack of historical records. In the present study we applied molecular, paleolimnological (trace metal, Itrax-µ-XRF and hyperspectral scanning, chronology), paleobotanical (pollen) and historical data to reconstruct cyanobacterial abundance and community composition and anthropogenic impacts in two dune lakes over a period of up to 1200 years. Metabarcoding and droplet digital PCR results showed very low levels of picocyanobacteria present in the lakes prior to about CE 1854 (1839-1870 CE) in the smaller shallow Lake Alice and CE 1970 (1963-1875 CE) in the larger deeper Lake Wiritoa. Hereafter bloom-forming cyanobacteria were detected and increased notably in abundance post CE 1984 (1982-1985 CE) in Lake Alice and CE 1997 (1990-2007 CE) in Lake Wiritoa. Currently, the magnitude of blooms is more pronounced in Lake Wiritoa, potentially attributable to hypoxia-induced release of phosphorus from sediment, introducing an additional source of nutrients. Generalized linear modelling was used to investigate the contribution of nutrients (proxy = bacterial functions), temperature, redox conditions (Mn:Fe), and erosion (Ti:Inc) in driving the abundance of cyanobacteria (ddPCR). In Lake Alice nutrients and erosion had a statistically significant effect, while in Lake Wiritoa nutrients and redox conditions were significant.

An integrative approach to assess the impact of disturbance on native fish in lakes.

Freshwater fish are in a perilous state with more than 30% of species considered critically endangered. Yet significant ecological and methodological complexities constrain our ability to determine how disturbances are impacting native fish communities. We review current methods used to assess the responses of fish communities, especially native fish, to disturbances, with a focus on lakes. These methods include contemporary population surveys, manipulative experimental approaches, paleolimnological approaches and Indigenous Knowledge and social histories. We identify knowledge gaps, such as a lack of baseline data for native fish, an inability to assess the impact of historical disturbances, stressor response dynamics in contemporary multi-stressor environments, and natural disturbance regimes. Our assessment of the current methods highlights challenges to filling these knowledge gaps using the reviewed methods. We advocate strongly for the implementation of an integrative approach that combines emerging technologies (i.e. molecular-based techniques in contemporary surveys and paleolimnology) and underutilised knowledge streams (i.e. Indigenous Knowledge and social histories) which should be used in concert with conventional methods. This integrative approach will allow researchers to determine the key drivers of decline and the degree of change, which will enable more informed and successful management actions.

A taxonomy-free diatom eDNA-based technique for assessing lake trophic level using lake sediments.

Anthropogenic eutrophication is one of the most pressing issues facing lakes globally. Our ability to manage lake eutrophication is hampered by the limited spatial and temporal extents of monitoring records, stemming from the time-consuming and expensive nature of physiochemical and biological monitoring. Diatom-based biomonitoring presents an alternative to traditional eutrophication monitoring, yet it is restricted by the high degree of taxonomic expertise required. Environmental DNA metabarcoding, while providing a promising substitute for diatom community enumeration, is plagued by inadequate taxonomic coverage of reference databases and methodological bias, limiting its use for biomonitoring. Here we show that taxonomy-free diatom-biomonitoring, in which environmental DNA metabarcoding data is utilised but not assigned to specific taxonomic classes, presents an accurate, fast, and relatively automated alternative to taxonomically assigned eutrophication biomonitoring. Our taxonomy-free index accounted for 85% of trophic level variability across 89 lakes and had the lowest average prediction error of the three approaches tested. By not relying on taxonomic identification or metabarcoding reference databases, taxonomy-free biomonitoring maintains diatom diversity that is lost in taxonomic assignment using molecular approaches. Furthermore, by utilising lake sediments, the approach outlined here presents a time-integrated estimation of lake trophic level and thus does not require time-consuming seasonal sampling. Taxonomy-free biomonitoring addresses the limitations of traditional physicochemical eutrophication monitoring and taxonomic biomonitoring alternatives and can be used to extend the spatial and temporal extents of eutrophication monitoring.

Characterizing carotenoids in cyanobacterial cultures - Opportunities and implications for paleolimnological studies.

Cyanobacterial blooms are increasing in frequency and intensity globally, impacting lake ecosystem health and posing a risk to human and animal health due to the toxins they can produce. Cyanobacterial pigments preserved in lake sediments provide a useful means of understanding the changes that have led to cyanobacterial blooms in lakes. However, there is some uncertainty as to whether specific carotenoids are unique to certain genera or types of cyanobacteria. To fill this knowledge gap, we analyzed pigments in 34 cyanobacteria cultures and applied the findings to sediments from three New Zealand lakes. The cyanobacterial carotenoids canthaxanthin, echinenone and zeaxanthin were detected in all cultures, whereas myxoxanthophyll was only detected in ten cultures (Microcoleus, Planktothrix and the picocyanobacteria cultures; Synechococcaceae). The sum of the individual carotenoid concentrations provided the strongest relationship with cyanobacterial biomass (R2 = 0.58) and could be used in paleolimnology studies to evaluate general cyanobacterial abundance. Ratios of canthaxanthin, zeaxanthin and myxoxanthophyll relative to echinenone indicated that carotenoid ratios could be used to differentiate picocyanobacteria and bloom-forming cyanobacteria, to some degree. High zeaxanthin/echinenone ratios were measured in picocyanobacteria and low zeaxanthin/echinenone ratios were measured in bloom-forming cyanobacteria. The zeaxanthin/echinenone ratio was applied to sediment core samples where the cyanobacterial community was also evaluated by 16S rRNA gene metabarcoding, with the zeaxanthin/echinenone ratios showing similar patterns to those observed in the cultures. The preliminary assessment described here suggests that zeaxanthin/echinenone ratios could provide a valuable paleoecological proxy for evaluating historical shifts in cyanobacterial communities and warrants further exploration.

Paleo-diatom records reveal ecological change not detected using traditional measures of lake eutrophication.

Lakes provide crucial ecosystem services and harbour unique and rich biodiversity, yet despite decades of research and management focus, cultural eutrophication remains a predominant threat to their health. Our ability to manage lake eutrophication is restricted by the lack of long-term monitoring records. To circumvent this, we developed a bio-indicator approach for inferring trophic level from lake diatom communities and applied this to sediment cores from two lakes experiencing eutrophication stress. Diatom indicators strongly predicted observed trophic levels, and when applied to sediment cores, diatom predicted trophic level reconstructions were consistent with monitoring data and land-use histories. However, there were significant recent shifts in diatom communities not captured by the diatom-based index or monitoring data, suggesting that conventional trophic level indices obscure important ecological change. New approaches, such as the one in this study, are critical to detect early changes in water quality and prevent the decline of lake ecosystems worldwide.

Resolving 500 Years of Anthropogenic Impacts in a Mesotrophic Lake: Nutrients Outweigh Other Drivers of Lake Change.

Interactions among multiple stressors, legacies of past perturbations, and the lack of historical information make it difficult to determine the influence of individual anthropogenic impacts on lakes and separate them from natural ecosystem variability. In the present study, we coupled paleolimnological approaches, historical data, and ecological experiments to disentangle the impacts of multiple long-term stressors on lake ecosystem structure and function. We found that the lake structure and function remained resistant to the impacts of catchment deforestation and erosion, and the introduction of several exotic fish species. Changes in ecosystem structure and function were consistent, with nutrient enrichment being the primary driver of change. Significant and sustained changes in the lake diatom community structure (and their nutrient requirements), bacterial community function, and paleolimnological proxies of ecosystem function coincided with nitrogen and phosphorus fertilizers in the catchment. The results highlight that the effects of increased nutrient inputs are much stronger than the influence of other, potentially significant, drivers of ecosystem change, and that the degree of nutrient impact can be underestimated by environmental monitoring due to its diffuse and accumulative nature. Delineating the effects of multiple anthropogenic drivers requires long-term records of both impacts and lake ecosystem change across multiple trophic levels.

Human access and deterministic processes play a major role in structuring planktonic and sedimentary bacterial and eukaryotic communities in lakes.

Lakes provide habitat for a diverse array of species and offer a wide range of ecosystem services for humanity. However, they are highly vulnerable as they are not only impacted by adverse actions directly affecting them, but also those on the surrounding environment. Improving knowledge on the processes responsible for community assembly in different biotic components will aid in the protection and restoration of lakes. Studies to date suggested a combination of deterministic (where biotic/abiotic factors act on fitness differences amongst taxa) and stochastic (where dispersal plays a larger factor in community assembly) processes are responsible for structuring biotic communities, but there is no consensus on the relative roles these processes play, and data is lacking for lakes. In the present study, we sampled different biotic components in 34 lakes located on the South Island of New Zealand. To obtain a holistic view of assembly processes in lakes we used metabarcoding to investigate bacteria in the sediment and surface waters, and eukaryotes in the sediment and two different size fractions of the water column. Physicochemical parameters were collected in parallel. Results showed that deterministic processes dominated the assembly of lake communities although the relative importance of variable and homogeneous selection differed among the biotic components. Variable selection was more important in the sediment (SSbact and SSeuks) and for the bacterioplankton (Pbact) while the assembly of the eukaryotic plankton (SPeuks, LPeuks) was driven more by homogeneous selection. The ease of human access to the lakes had a significant effect on lake communities. In particular, clade III of SAR11 and Daphnia pulex were only present in lakes with public access. This study provides insights into the distribution patterns of different biotic components and highlights the value in understanding the drivers of different biological communities within lakes.

Using metabarcoding and droplet digital PCR to investigate drivers of historical shifts in cyanobacteria from six contrasting lakes.

The frequency and intensity of cyanobacterial blooms is increasing worldwide. Multiple factors are implicated, most of which are anthropogenic. New Zealand provides a useful location to study the impacts of human settlement on lake ecosystems. The first humans (Polynesians) arrived about 750 years ago. Following their settlement, there were marked landscape modifications which intensified after European settlement about 150 years ago. The aims of this study were to reconstruct cyanobacterial communities in six lakes over the last 1000 years and explore key drivers of change. Cyanobacterial environmental DNA was extracted from sediment cores and analysed using metabarcoding and droplet digital PCR. Cyanobacteria, including potentially toxic or bloom forming species, were already present in these lakes prior to human arrival, however their overall abundance was low. Total cyanobacteria abundance and richness increased in all lakes after European settlement but was very pronounced in four lakes, where bloom-forming taxa became dominant. These shifts occurred concomitant with land-use change. The catchment of one deteriorated lake is only moderately modified, thus the introduction of non-native fish is posited as the key factor driving this change. The paleolimnological approach used in this study has enabled new insights into timing and potential causes of changes in cyanobacterial communities.

Molecular and Pigment Analyses Provide Comparative Results When Reconstructing Historic Cyanobacterial Abundances from Lake Sediment Cores.

Understanding the historical onset of cyanobacterial blooms in freshwater bodies can help identify their potential drivers. Lake sediments are historical archives, containing information on what has occurred in and around lakes over time. Paleolimnology explores these records using a variety of techniques, but choosing the most appropriate method can be challenging. We compared results obtained from a droplet digital PCR assay targeting a cyanobacterial-specific region of the 16S rRNA gene in sedimentary DNA and cyanobacterial pigments (canthaxanthin, echinenone, myxoxanthophyll and zeaxanthin) analysed using high-performance liquid chromatography in four sediment cores. There were strong positive relationships between the 16S rRNA gene copy concentrations and individual pigment concentrations, but relationships differed among lakes and sediment core depths within lakes. The relationships were more consistent when all pigments were summed, which we attribute to different cyanobacteria species, in different lakes, at different times producing different suites of pigments. Each method had benefits and limitations, which should be taken into consideration during method selection and when interpreting paleolimnological data. We recommend this biphasic approach when making inferences about changes in the entire cyanobacterial community because they yielded complementary information. Our results support the view that molecular methods can yield results similar to traditional paleolimnological proxies when caveats are adequately addressed.

Deciphering the molecular signal from past and alive bacterial communities in aquatic sedimentary archives.

Lake sediments accumulate information on biological communities thus acting as natural archives. Traditionally paleolimnology has focussed on fossilized remains of organisms, however, many organisms do not leave fossil evidence, meaning major ecosystem components are missing from environmental reconstructions. Many paleolimnology studies now incorporate molecular methods, including investigating microbial communities using environmental DNA (eDNA), but there is uncertainty about the contribution of living organisms to molecular inventories. In the present study, we obtained DNA and RNA inventories from sediment spanning 700 years to investigate the contribution of past and active communities to the molecular signal from sedimentary archives. Additionally, a droplet digital PCR (ddPCR) targeting the 16S ribosomal RNA (16S rRNA) gene of the photosynthetic cyanobacterial genera Microcystis was used to explore if RNA signals were from legacy RNA. We posit that the RNA signal is a mixture of legacy RNA, dormant cells, living bacteria and modern-day trace level contaminants that were introduced during sampling and preferentially amplified. The presence of legacy RNA was confirmed by the detection of Microcystis in sediments aged to ~200 years ago. Recent comparisons between 16S rRNA gene metabarcoding and traditional paleo proxies showed that past changes in bacterial communities can be reconstructed from sedimentary archives. The recovery of RNA in the present study has provided new insights into the origin of these signals. However, caution is required during analysis and interpretation of 16S rRNA gene metabarcoding data especially in recent sediments were there are potentially active bacteria.

A bacterial index to estimate lake trophic level: National scale validation.

Lakes and their catchments have been subjected to centuries to millennia of exploitation by humans. Efficient monitoring methods are required to promote proactive protection and management. Traditional monitoring is time consuming and expensive, which limits the number of lakes monitored. Lake surface sediments provide a temporally integrated representation of environmental conditions and contain high microbial biomass. Based on these attributes, we hypothesized that bacteria associated with lake trophic states could be identified and used to develop an index that would not be confounded by non-nutrient stressor gradients. Metabarcoding (16S rRNA gene) was used to assess bacterial communities present in surface sediments from 259 non-saline lakes in New Zealand encompassing a range of trophic states from alpine microtrophic lakes to lowland hypertrophic lakes. A subset of lakes (n = 96) with monitoring data was used to identify indicator amplicon sequence variants (ASVs) associated with different trophic states. A total of 10,888 indicator taxa were identified and used to develop a Sediment Bacterial Trophic Index (SBTI), which signficantly correlated (r2 = 0.842, P < 0.001) with the Trophic Lake Index. The SBTI was then derived for the remaining 163 lakes, providing new knowledge of the trophic state of these unmonitored lakes. This new, robust DNA-based tool provides a rapid and cost-effective method that will allow a greater number of lakes to be monitored and more effectively managed in New Zealand and globally. The SBTI could also be applied in a paleolimnological context to investigate changes in trophic status over centuries to millennia.

Development of droplet digital Polymerase Chain Reaction assays for the detection of long-finned (Anguilla dieffenbachii) and short-finned (Anguilla australis) eels in environmental samples.

Freshwater eels are ecologically, and culturally important worldwide. The New Zealand long-finned eel (Anguilla dieffenbachii) and short-finned eel (Anguilla australis) are apex predators, playing an important role in ecosystem functioning of rivers and lakes. Recently, there has been a national decline in their populations due to habitat destruction and commercial harvest. The emergence of targeted environmental DNA detection methodologies provides an opportunity to enhance information about their past and present distributions. In this study we successfully developed species-specific droplet digital Polymerase Chain Reaction (ddPCR) assays to detect A. dieffenbachii and A. australis DNA in water and sediment samples. Assays utilized primers and probes designed for regions of the mitochondrial cytochrome b and 16S ribosomal RNA genes in A. dieffenbachii and A. australis, respectively. River water samples (n = 27) were analyzed using metabarcoding of fish taxa and were compared with the ddPCR assays. The presence of A. dieffenbachii and A. australis DNA was detected in a greater number of water samples using ddPCR in comparison to metabarcoding. There was a strong and positive correlation between gene copies (ddPCR analyses) and relative eel sequence reads (metabarcoding analyses) when compared to eel biomass. These ddPCR assays provide a new method for assessing spatial distributions of A. dieffenbachii and A. australis in a range of environments and sample types.

Investigating variability in microbial community composition in replicate environmental DNA samples down lake sediment cores.

Lake sediments are natural archives that accumulate information on biological communities and their surrounding catchments. Paleolimnology has traditionally focussed on identifying fossilized organisms to reconstruct past environments. In the last decade, the application of molecular methodologies has increased in paleolimnological studies, but further research investigating factors such as sample heterogeneity and DNA degradation are required. In the present study we investigated bacterial community heterogeneity (16S rRNA metabarcoding) within depth slices (1-cm width). Sediment cores were collected from three lakes with differing sediment compositions. Samples were collected from a variety of depths which represent a period of time of approximately 1,200 years. Triplicate samples were collected from each depth slice and bacterial 16S rRNA metabarcoding was undertaken on each sample. Accumulation curves demonstrated that except for the deepest (oldest) slices, the combination of three replicate samples were insufficient to characterise the entire bacterial diversity. However, shared Amplicon Sequence Variants (ASVs) accounted for the majority of the reads in each depth slice (max. shared proportional read abundance 96%, 86%, 65% in the three lakes). Replicates within a depth slice generally clustered together in the Non-metric multidimensional scaling analysis. There was high community dissimilarity in older sediment in one of the cores, which was likely due to the laminae in the sediment core not being horizontal. Given that most paleolimnology studies explore broad scale shifts in community structure rather than seeking to identify rare species, this study demonstrates that a single sample is adequate to characterise shifts in dominant bacterial ASVs.

The Role of Environmental Processes and Geographic Distance in Regulating Local and Regionally Abundant and Rare Bacterioplankton in Lakes.

Bacteria are vital components of lake systems, driving a variety of biogeochemical cycles and ecosystem services. Bacterial communities have been shown to have a skewed distribution with a few abundant species and a large number of rare species. The contribution of environmental processes or geographic distance in structuring these components is uncertain. The discrete nature of lakes provides an ideal test case to investigate microbial biogeographical patterns. In the present study, we used 16S rRNA gene metabarcoding to examine the distribution patterns on local and regional scales of abundant and rare planktonic bacteria across 167 New Zealand lakes covering broad environmental gradients. Only a few amplicon sequence variants (ASVs) were abundant with a higher proportion of rare ASVs. The proportion of locally abundant ASVs was negatively correlated with the percentage of high productivity grassland in the catchment and positively with altitude. Regionally rare ASVs had a restricted distribution and were only found in one or a few lakes. In general, regionally abundant ASVs had higher occupancy rates, although there were some with restricted occupancy. Environmental processes made a higher contribution to structuring the regionally abundant community, while geographic distances were more important for regionally rare ASVs. A better understanding of the processes structuring the abundance and distribution of bacterial communities within lakes will assist in understand microbial biogeography and in predicting how these communities might shift with environmental change.

Long-term patterns of hillslope erosion by earthquake-induced landslides shape mountain landscapes.

Widespread triggering of landslides by large storms or earthquakes is a dominant mechanism of erosion in mountain landscapes. If landslides occur repeatedly in particular locations within a mountain range, then they will dominate the landscape evolution of that section and could leave a fingerprint in the topography. Here, we track erosion provenance using a novel combination of the isotopic and molecular composition of organic matter deposited in Lake Paringa, New Zealand. We find that the erosion provenance has shifted markedly after four large earthquakes over 1000 years. Postseismic periods eroded organic matter from a median elevation of 722 +329/-293 m and supplied 43% of the sediment in the core, while interseismic periods sourced from lower elevations (459 +256/-226 m). These results are the first demonstration that repeated large earthquakes can consistently focus erosion at high elevations, while interseismic periods appear less effective at modifying the highest parts of the topography.

Local factors drive bacterial and microeukaryotic community composition in lake surface sediment collected across an altitudinal gradient.

Lake surface sediments are dominated by microorganisms that play significant roles in biogeochemical cycling within lakes. There is limited knowledge on the relative importance of local environmental factors and altitude on bacterial and microeukaryotic community richness and composition in lake sediments. In the present study, surface sediment samples were collected from 40 lakes along an altitude gradient (2-1215 m). Microbial communities were characterized using 16S (bacteria) and 18S (microeukaryotes) rRNA gene metabarcoding. Bacterial and microeukaryotic richness were not correlated with altitude but instead to environmental variables (e.g. area of water in the catchment (bacteria: R = -0.43). For both bacteria and microeukaryotes, dissimilarity in the community structure had a higher correlation to combined environmental variables (without altitude) (bacteria: R = 0.53; microeukaryotes: R = 0.55) than altitude alone (bacteria: R = 0.34; microeukaryotes: R = 0.47). Sediment sulfur and productive grassland were important variables in determining the relative abundance of sulfate reducing bacteria. Nitrospira, was positively related to altitude but negatively to water column total organic carbon and the proportion of productive grassland in the catchment. Little overlap in amplicon sequence variants was shown amongst lakes. This has important considerations for management decisions, suggesting that to protect biodiversity, conservation of numerous lakes and lake types is required.

Earthquakes drive large-scale submarine canyon development and sediment supply to deep-ocean basins.

Although the global flux of sediment and carbon from land to the coastal ocean is well known, the volume of material that reaches the deep ocean-the ultimate sink-and the mechanisms by which it is transferred are poorly documented. Using a globally unique data set of repeat seafloor measurements and samples, we show that the moment magnitude (Mw) 7.8 November 2016 Kaikoura earthquake (New Zealand) triggered widespread landslides in a submarine canyon, causing a powerful "canyon flushing" event and turbidity current that traveled >680 km along one of the world's longest deep-sea channels. These observations provide the first quantification of seafloor landscape change and large-scale sediment transport associated with an earthquake-triggered full canyon flushing event. The calculated interevent time of ~140 years indicates a canyon incision rate of 40 mm year-1, substantially higher than that of most terrestrial rivers, while synchronously transferring large volumes of sediment [850 metric megatons (Mt)] and organic carbon (7 Mt) to the deep ocean. These observations demonstrate that earthquake-triggered canyon flushing is a primary driver of submarine canyon development and material transfer from active continental margins to the deep ocean.