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Lara, E.

Publications and source records attributed to Lara, E..

At least 19 recordsLinked to original sources

Tropical Peatlands as Hotspots of Hidden Arcellinida Diversity: Insights from Maludam National Park, Borneo (Sarawak, Malaysia)

Recent investigations on tropical peatlands have shown their extremely high efficiency as carbon sinks, far beyond better studied boreal bogs. The microbial diversity found in those ecosystems, as well as their corresponding ecological and metabolic processes, are just starting to be revealed, and differ considerably from high latitudes systems. However, these efforts have been concentrated almost exclusively towards prokaryotes and fungi, leaving protist communities virtually unknown. Arcellinida (testate amoebae) are well acknowledged to play an important role in boreal and temperate peatland ecology by driving microbial community composition as top predators, but also as nutrient recyclers and carbon fixers. Yet, to our knowledge, no community-level study has addressed their diversity and its drivers in tropical peatlands. In this study, we surveyed Arcellinida communities' diversity across five functionally distinct vegetation-hydrological zones situated along a hydrological transect in a coastal tropical peatland in the Maludam National Park, Malaysia. Taxonomic composition of Arcellinida communities, characterized by a high diversity of Trigonopyxis-related taxa, indicated fundamental differences in diversity and community composition from extratropical ecosystems. Species richness was significantly higher in the zones with developed understory vegetation. Furthermore, the composition of diversity varied significantly among vegetation-hydrological zones. Altogether, this study shows unique Arcellinida communities in a Southeastern Asian peatland and suggests the importance of vegetation-hydrological characteristics for protist diversity.

ecology↗

Less diverse but uniquely composed: Canopy cover and elevation shape Neotropical Arcellinida assemblages in the Peruvian Andes

The Peruvian Andes, and especially its slopes towards the Amazonian Basin are well-known biodiversity hotspots for plants and animals, which represent the visible part of biodiversity. However, not much is known about microeukaryotic diversity, despite the significant role protists play in the ecosystem. Based on a molecular survey of the diversity of Arcellinida (a group of amoebae well known for its environmental sensitivity and restricted geographical distributions), we estimated its diversity along an elevation gradient spanning more than 3000 meters. Surprisingly, Neotropical forest Arcellinida were less abundant and diverse than in most other extratropical biomes; nevertheless, communities differed strongly in their taxonomic composition as compared with forests in higher latitudes. Notably, sequences related to Trigonopyxis were most abundant, whereas this group occurs only marginally in extratropical settings. Elevation influenced beta diversity, showing different assembly processes occurring at different elevation belts. Canopy cover positively influenced both phylogenetic and taxonomic diversity, showing the importance of above and below ground integration in tropical forests.

ecology↗

Precipitation seasonality and soil carbon shape Arcellinida diversity across Bulgaria's ecosystems

Recent years have witnessed the discovery of an immense diversity of protists in almost all ecosystems on Earth. However, a major challenge consists in identifying diversity patterns and the drivers that explain the distribution of this diversity. While several works have focused on local parameters, only a few studies have addressed this question at regional scales. In this study, we characterize the diversity and distribution of Arcellinida (testate amoebae) at regional scale of a climatically contrasted country, Bulgaria. We collected 75 samples geographically evenly distributed and representative of the country's natural ecosystems and used metabarcoding to determine their diversity. Communities composition differed markedly among habitats, revealing sharp beta diversity patterns. "Mesophilic" environments such as forests and ponds were most diverse, while acidic and oligotrophic Sphagnum peatlands were poorest and hosted the highest proportion of unique taxa. Our analyses of richness-environment based on a Random Forest approach applied to all soil systems indicated that stable humidity (low silt content, low precipitation seasonality) and intermediate soil carbon stocks had a positive effect on richness. The predicted increase in continentality in the next decades in Bulgaria calls for testing climatic models to evaluate the potential risk of declining Arcellinida biodiversity.

ecology↗

Profiling of Aggregated Amyloid β42-induced Proteomic Alterations in KOLF2.1J Induced Pluripotent Stem Cell-derived Neurons

Amyloid[beta]; (A[beta];) plaques are a hallmark of Alzheimer[prime]s disease (AD). A human cellular neuronal model that recaptures A[beta];-induced pathology is critical for advancing AD research. However, comprehensive proteomic profiling of A[beta];-induced cellular model remains elusive. In this study, we investigated the proteomic changes in induced pluripotent stem cell (iPSC)-derived neurons (iNs) exposed to synthetic A[beta]; (1-42) peptides (A[beta];42A) to improve our understanding of the cellular responses of A[beta]; aggregates and to establish a human-related platform for AD research. A[beta];42A formed extracellular aggregates around neuronal soma and neurites, impaired neurite outgrowth, and induced the expression of multiple AD-associated genes, such as APOE, BACE1, ADAM10. To define the molecular landscape of A[beta];-induced neuronal dysfunction, we performed proteomic analyses of whole-cell lysates as well as soma- and neurite-enriched fractions. Proteomic profiling revealed extensive gene alterations in pathways associated with synaptic function, neuronal maintenance, and AD pathogenesis, such as the upregulation of APOE. Importantly, the A[beta];42A-iNs system recapitulated molecular signatures observed in AD brain tissue and cerebrospinal fluid. Representative protein changes, including APOE upregulation and its colocalization with A[beta]; aggregates, were further validated in postmortem human AD brain tissue. Together, these findings manifest that the A[beta];42A-iNs system reproduces multiple cellular and molecular features of AD and exhibits strongly consensus with clinical observations. It provides a valuable platform for investigating A[beta];-driven neurodegeneration and for the discovery of therapeutic targets for AD.

neuroscience↗

Threshold responses of aquatic microbial diversity to terrestrial land use across Mediterranean catchments

Climate driven aridification and intensified human activity are placing increasing pressure on Mediterranean freshwater ecosystems. These impacts propagate from land to water, altering nutrient regimes and reshaping aquatic microbial communities. We analysed Arcellinida diversity across 363 lentic inland saline and freshwater sediment samples spanning broad gradients of land use, water chemistry, soil properties, and climate in southern Spain. Random forest models identified terrestrial land use intensity followed by water chemistry as main predictors of community diversity. Diversity declined sharply in sites with population densities above [~]33 inhabitants/km{superscript 2} and under eutrophic conditions, but peaked in oligotrophic systems with stable, carbon rich soils. These threshold responses demonstrate that aquatic protist assemblages integrate both long term terrestrial pressures and current water conditions. Overall, our findings show that landscape transformation and its cascading effects on water quality dominate community assembly, and that the combination of community level diversity metrics with selected taxon-level indicators capture ecosystem degradation more consistently than relying on a single metric.

ecology↗

Widespread decoupling between abundance and genetic diversity, and strong local genetic structuring in marine unicellular eukaryotes

How abundance, connectivity, and genetic diversity covary across space remains a central question in evolutionary biology. Using a global environmental DNA metabarcoding dataset targeting mitochondrial cytochrome c oxidase subunit I (COI), we examined relationships among metabarcoding-derived abundance, nucleotide diversity, and spatial genetic connectivity across marine eukaryotes. We found a widespread but incomplete decoupling between abundance and genetic diversity, particularly in unicellular lineages. By contrast, distance-decay patterns in abundance, diversity, and connectivity were broadly similar across eukaryotes and more strongly associated with cellularity. Focusing on the Iberian Peninsula, we reconstructed locality-centred haplotype networks to quantify the spatial organization of intraspecific genetic variation. Unicellular lineages showed stronger local genetic structuring, whereas multicellular organisms exhibited clearer large-scale biogeographic partitioning. Overall, our results reveal shared biogeographic organization across eukaryotes while demonstrating that abundance-based biodiversity metrics alone cannot fully capture evolutionary and demographic processes, especially in microbial organisms.

ecology↗

The complete genome of the KOLF2.1J reference iPSC line

While induced pluripotent stem cells (iPSCs) have gained popularity in studying neurodegenerative diseases, the heterogeneity of stem cells used across studies impacts cross-study comparison. The iPSC Neurodegenerative Disease Initiative (iNDI) selected the KOLF2.1J cell line and prioritized its use as a reference standard for studying the effects of pathogenic variants on cell biology due to its stability and neutral neurodegenerative disease genetic risk. This cell line, and its derivatives expressing over 100 variants related to Alzheimers disease, Parkinsons disease, and other neurological diseases, are available for academic and industry access. Current genomic data analyses are limited by the use of a human reference genome that does not capture the complete genetic background of a given iPSC line. While in the future this issue may be partially mitigated by the creation of a comprehensive human pangenome, previous work has shown that generating custom genomes is of value both to characterize the variation present and to serve as a more appropriate genomic reference. Here, we generated and characterized a custom complete genome assembly from KOLF2.1J. Mapping of sequencing reads to a personalized diploid assembly results in more comprehensive mapping compared to traditional linear references (i.e GRCh38). In addition, we provide a comprehensive custom gene annotation along with isoform expression and differential methylation analyses across multiple cell types. The assembly and all additional data is browsable and publicly available. This resource will enable more accurate investigation of the KOLF2.1J cell line and any genomics data generated compared to using traditional generalized references, while also serving as a foundational approach for establishing custom reference assemblies for other high-value iPSC lines.

genomics↗

Quaternary ice ages shaped protists phylogeography: the case of Arcellinida in the Iberian Peninsula

The Quaternary glaciations profoundly shaped the biogeography of plants and animals, yet their impact on microbial eukaryotes remains largely unexplored. We tested the "genetic legacy of the Quaternary" (GLQ) paradigm in terrestrial protists using Arcellinida testate amoebae diversity distribution across the Iberian Peninsula, a well-established glacial refugium. To do so, we compiled the most extensive Arcellinida metabarcoding dataset to date (ArKOI), including 615 samples from multiple continents and ecosystems. Our results showed that hotspots of intra-OTU genetic diversity align with known Iberian refugia, supporting the concept of "refugia within refugia", and displaying clear ecoregion-specific climatic niches. Patterns of spatial clustering, niche breadth, and historical demographic reconstruction revealed repeated range contractions and expansions during the Pleistocene, mirroring those observed in macro-organisms. These findings extend the GLQ paradigm to protists, highlighting shared historical and ecological processes across the eukaryotic tree of life and contributing to a unified theory of biogeographic responses to climatic change.

ecology↗

Dissecting the biological impact of GBA1 mutations using multi-omics in an isogenic setting

GBA1 is a risk gene for multiple neurodegenerative diseases, including Lewy Body Dementia and Parkinsons disease, and biallelic pathogenic variants in the gene result in the lysosomal storage disorder Gaucher disease. GBA1 encodes the enzyme glucocerebrosidase (GCase), and alterations in the gene result in reduced enzymatic activity, which affects lysosome function downstream. Induced pluripotent stem cells (iPSCs) are a useful tool for testing the functional consequences of gene variants in an isogenic setting. Additionally, they can be used to perform multiomic studies to explore biological effects independent of disease mechanisms. Using CRISPR-edited isogenic KOLF2.1J iPSC lines containing pathogenic GBA1 variants D409H (p.D448H), D409V (p.D448V) and GBA1 knockout line generated by the iPSC Neurodegenerative Disease Initiative (iNDI), we examined potential molecular mechanisms and downstream consequences of GCase reduction. In this study, we confirm that this isogenic series behaves as expected for loss of function variants, despite the known difficulties with GBA1 editing. We identified that there are limited overlapping results across cell types suggesting potential different downstream effects caused by GBA1 variants. Additionally, we note that RNA-based quantitation may not be the best method to characterize GCase mechanisms, but protein and metabolomic analyses may be used to evaluate differences across genotypes.

genomics↗

Experimental suppression of a keystone protist triggers mesopredator release and biotic homogenization in complex soil microbial communities

The keystone species concept suggests that certain members of an ecological community, despite their low abundance, exert disproportionately large effects on species diversity and composition. In microbial ecology, experimental validation of this concept is limited due to significant technical challenges associated with selective species manipulation. Here, we tested this concept within a soil microbial food web by selectively suppressing a protist predator using phototoxicity induced by excessive excitation light during fluorescence microscopy within a microfluidic soil chip system. We targeted a Hypotrichia ciliate taxon--presumed primarily bacterivorous under our experimental conditions--and combined microscopy with metabarcoding of multiple microbial trophic levels to evaluate the effects of this suppression on microbial community abundance, diversity, and composition. Over the 20-day incubation, the chip system supported complex communities of bacteria, fungi, and protists. Following Hypotrichia suppression, two distinct ecological responses were observed: first, an increase in flagellate abundance that was consistent with mesopredator release and accompanied a significant rise in overall protist diversity; second, a convergence in protist community composition, indicative of biotic homogenization. Surprisingly, bacterial community abundance, richness, and composition remained unaffected, likely due to compensatory predation by increased numbers of bacterivorous flagellates. In contrast, fungal diversity decreased following Hypotrichia suppression, presumably resulting from the altered protist communities that favored facultative fungal consumers. Collectively, these findings provide direct experimental evidence that low-abundance microbial predators can function as keystone species, modulating predator community composition and diversity and having cascading effects on lower trophic levels within the brown microbial food web.

ecology↗

Temporal dynamics of proteome and phosphorproteome during neuronal differentiation in the reference KOLF2.1J iPSC line

Induced pluripotent stem cell (iPSC)-derived neurons have emerged as a powerful model to investigate both neuronal development and neurodegenerative diseases. Although transcriptomics and imaging have been applied to characterize neuronal development signatures, comprehensive datasets of protein and post-translational modifications (PTMs) are not readily available. Here, we applied quantitative proteomics and phosphoproteomics to profile the differentiation of the KOLF2.1J iPSC line, the first reference line of the iPSC Neurodegenerative Disease Initiative (iNDI) project. We developed an automated workflow enabling high-coverage enrichment of proteins and phosphoproteins. Our results revealed molecular signatures across proteomic and phosphoproteomic landscapes during differentiation of iPSC-derived neurons. Proteomic data highlighted distinct changes in mitochondrial pathways throughout the course of differentiation, while phosphoproteomics revealed specific regulatory dynamics in GTPase signaling pathways and microtubule proteins. Additionally, phosphosite dynamics exhibited discordant trends compared to protein expression, particularly in processes related to axon functions and RNA transport. Furthermore, we mapped the kinase dynamic changes that are critical for neuronal development and maturation. We developed an interactive Web app (https://niacard.shinyapps.io/Phosphoproteome/) to visualize temporal landscape dynamics of protein and phosphosite expression. By establishing baselines of proteomic and phosphoproteomic profiles for neuronal differentiation, this dataset offers a valuable resource for future research into neuronal development and neurodegenerative diseases using this reference iPSC line. HighlightsO_LITemporal dynamics of proteome and phosphoproteome profiles in KOLF2.1J iPSC derived neurons. C_LIO_LIPhosphoproteomics highlights GTPase signaling and microtubule regulation in neuronal differentiation. C_LIO_LIKinome mapping reveals a shift in kinase activity patterns from early to late differentiation. C_LIO_LIShinyapp for visualizing the trajectory of protein and phosphosite expression during neuronal differentiation. C_LI

neuroscience↗

iPSC Motor Neurons with Familial ALS Mutations Capture Gene Expression Changes in Postmortem Sporadic ALS Motor Neurons

Motor neuron degeneration, the defining feature of ALS, is a primary example of cell-type specificity in neurodegenerative diseases. Using isogenic pairs of iPSCs harboring different familial ALS mutations, we assess the capacity of iPSC-derived spinal motor neurons, sensory neurons, astrocytes, and superficial cortical neurons to capture disease features including transcriptional and splicing dysregulation observed in human post-mortem neurons. At surprisingly early time points, differentially regulated genes in iPSC-derived spinal motor neurons, but not other cell types, overlap with one-third of the differentially regulated genes in laser-dissected motor neurons from postmortem spinal cords. The extent of dysregulation correlates well between iPSC-derived and bona fide spinal motor neurons. In iPSC-derived spinal motor neurons, but not other derived cell types, we detect downregulation of genes affected by TDP-43-dependent aberrant splicing. This reduction takes place exclusively within genotypes known to involve TDP-43 pathology and occurs without evidence of TDP-43 mislocalization or protein level alteration.

neuroscience↗

A needle in a haystack: a new metabarcoding approach to survey diversity at the species level of Arcellinida (Amoebozoa: Tubulinea)

Environmental DNA-based diversity studies have increased in popularity with the development of high throughput sequencing technologies. This permits the potential simultaneous retrieval of vast amounts of molecular data from many different organisms and species, thus contributing to a wide range of biological disciplines. Environmental DNA protocols designed for protists often focused on the highly conserved small subunit of the ribosome gene, that does not permit species-level assignments. On the other hand, eDNA protocols aiming at species-level assignments allow a fine level ecological resolution and reproducible results. These protocols are currently applied to organisms living in marine and shallow lotic freshwater ecosystems, often in a bioindication purpose. Therefore, in this study, we present a species-level eDNA protocol, designed to explore diversity of Arcellinida (Amoebozoa: Tubulinea) testate amoebae taxa, that is based on mitochondrial cytochrome oxidase subunit I (COI). These organisms are widespread in lentic water bodies and soil ecosystems. We applied this protocol to 42 samples from peatlands, estuaries and soil environments, recovering all the infraorders in Glutinoconcha (with COI data), except for Hyalospheniformes. Our results revealed an unsuspected diversity in morphologically homogeneous groups such as Cylindrothecina, Excentrostoma or Sphaerothecina. With this protocol we expect to revolutionize the design of modern distributional Arcellinida surveys. Our approach involve a rapid and cost effective analysis of testate amoeba diversity living in contrasted ecosystems. Therefore, Arcellinida clade have the potential to be established as a model group for an array of theoretical and applied studies.

microbiology↗

Microbial landscape ecology: landscape structure better predicts soil protist diversity than local edaphic variables in alpine meadows

ContextHuman-induced changes in landscape structure are among the main causes of biodiversity loss. Despite their important contribution to biodiversity and ecosystem functioning, microbes - and particularly protists - remain spatially understudied. Soil microbiota are most often driven by local soil properties, but the influence of the surrounding landscape is rarely assessed. ObjectivesWe assessed the effect of landscape structure on soil protist alpha and beta diversity in meadows in the western Swiss Alps. MethodsWe sampled 178 plots along an elevation gradient representing a broad range of environmental conditions and land-use. We measured landscape structure around each plot at 5 successive spatial scales (i.e. neighbourhood windows of increasing radius, ranging from 100 to 2000 m around a plot). We investigated the changes of protist alpha and beta diversity as a function of landscape structure, local environmental conditions and geographic distance. ResultsLandscape structures played a key role for protist alpha and beta diversity. The percentage of meadows, forests, or open habitats had the highest influence among all landscape metrics. The importance of landscape structure was comparable to that of environmental conditions and spatial variables, and increased with the size of the neighbourhood window considered. ConclusionsOur results suggest that dispersal from neighbouring habitats is a key driver of protist alpha and beta diversity which highlight the importance of landscape-scale assembly mechanisms for microbial diversity. Landscape structure emerges as a key driver of microbial communities which has profound implications for our understanding of the consequences of land-use change on soil microbial communities and their associated functions.

ecology↗

A reference induced pluripotent stem cell line for large-scale collaborative studies

Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate iPSC lines and deeply characterised their genetic properties using whole genome sequencing, their genomic stability upon CRISPR/Cas9-based gene editing, and their phenotypic properties including differentiation to commonly-used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and hundreds of its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field. SummaryThe authors of this collaborative study deeply characterized human induced pluripotent stem cell (iPSC) lines to rationally select a clonally-derived cell line that performs well across multiple modalities. KOLF2.1J was identified as a candidate reference cell line based on single-cell analysis of its gene expression in the pluripotent state, whole genome sequencing, genomic stability after highly efficient CRISPR-mediated gene editing, integrity of the p53 pathway, and the efficiency with which it differentiated into multiple target cell populations. Since it is deeply characterized and can be readily acquired, KOLF2.1J is an attractive reference cell line for groups working with iPSCs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/472643v6_ufig1.gif" ALT="Figure 1"> View larger version (84K): org.highwire.dtl.DTLVardef@cef106org.highwire.dtl.DTLVardef@31d5d8org.highwire.dtl.DTLVardef@1ce6d8corg.highwire.dtl.DTLVardef@17a19d2_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

A fully automated FAIMS-DIA proteomic pipeline for high-throughput characterization of iPSC-derived neurons

Fully automated proteomic pipelines have the potential to achieve deep coverage of cellular proteomes with high throughput and scalability. However, it is important to evaluate performance, including both reproducibility and ability to provide meaningful levels of biological insight. Here, we present an approach combining high field asymmetric waveform ion mobility spectrometer (FAIMS) interface and data independent acquisition (DIA) proteomics approach developed as part of the induced pluripotent stem cell (iPSC) Neurodegenerative Disease Initiative (iNDI), a large-scale effort to understand how inherited diseases may manifest in neuronal cells. Our FAIMS-DIA approach identified more than 8000 proteins per mass spectrometry (MS) acquisition as well as superior total identification, reproducibility, and accuracy compared to other existing DIA methods. Next, we applied this approach to perform a longitudinal proteomic profiling of the differentiation of iPSC-derived neurons from the KOLF2.1J parental line used in iNDI. This analysis demonstrated a steady increase in expression of mature cortical neuron markers over the course of neuron differentiation. We validated the performance of our proteomics pipeline by comparing it to single cell RNA-Seq datasets obtained in parallel, confirming expression of key markers and cell type annotations. An interactive webapp of this temporal data is available for aligned-UMAP visualization and data browsing (https://share.streamlit.io/anant-droid/singlecellumap). In summary, we report an extensively optimized and validated proteomic pipeline that will be suitable for large-scale studies such as iNDI.

neuroscience↗

Human Prefoldin Modulates Co-transcriptional Pre-mRNA Splicing

Prefoldin is a heterohexameric complex conserved from archaea to humans that plays a cochaperone role during the co-translational folding of actin and tubulin monomers. Additional functions of prefoldin have been described, including a positive contribution to transcription elongation and chromatin dynamics in yeast. Here we show that prefoldin perturbations provoked transcriptional alterations across the human genome. Severe pre-mRNA splicing defects were also detected, particularly after serum stimulation. We found impairment of co-transcriptional splicing during transcription elongation, which explains why the induction of long genes with a high number of introns was affected the most. We detected genome-wide prefoldin binding to transcribed genes and found that it correlated with the negative impact of prefoldin depletion on gene expression. Lack of prefoldin caused global decrease in Ser2 and Ser5 phosphorylation of the RNA polymerase II carboxy-terminal domain. It also reduced the recruitment of the CTD kinase CDK9 to transcribed genes, and the association of splicing factors PRP19 and U2AF65 to chromatin, which is known to depend on CTD phosphorylation. Altogether the reported results indicate that human prefoldin is able to act locally on the genome to modulate gene expression by influencing phosphorylation of elongating RNA polymerase II, and thereby regulating co-transcriptional splicing.

molecular biology↗

A user guide to environmental protistology: primers, metabarcoding, sequencing, and analyses

Protists - all eukaryotes besides fungi, animals, and plants - represent a major part of the taxonomic and functional diversity of eukaryotic life on the planet and drive many ecosystem processes. However, knowledge of protist communities and their diversity lags behind that of most other groups of organisms, largely due to methodological constraints. While protist communities differ markedly between habitats and biomes, they can be studied in very similar ways. Here we provide a guide to current molecular approaches used for studying protist diversity, with a particular focus on amplicon-based high-throughput sequencing (metabarcoding). We highlight that the choice of suitable primers artificially alters community profiles observed in metabarcoding studies. While there are no true universal primers to target all protist taxa as a whole, we identify some primer combinations with a wide taxonomic coverage and provide detailed information on their properties. Although environmental protistan ecological research will probably shift towards PCR-free metagenomics or/and transcriptomic approaches in a near future, metabarcoding will remain the method of choice for in-depth community analyses and taxon inventories in biodiversity surveys and ecological studies, due its great cost-efficiency, sensitivity, and throughput. In this paper we provide a guide for scientists from a broad range of disciplines to implement protists in their ecological analyses.

microbiology↗