Exploring freshwater stream bacterial communities as indicators of land use intensity.

BACKGROUND: Stream ecosystems comprise complex interactions among biological communities and their physicochemical surroundings, contributing to their overall ecological health. Despite this, many monitoring programs ignore changes in the bacterial communities that are the base of food webs in streams, often focusing on stream physicochemical assessments or macroinvertebrate community diversity instead. We used 16S rRNA gene sequencing to assess bacterial community compositions within 600 New Zealand stream biofilm samples from 204 sites within a 6-week period (February-March 2010). Sites were either dominated by indigenous forests, exotic plantation forests, horticulture, or pastoral grasslands in the upstream catchment. We sought to predict each site's catchment land use and environmental conditions based on the composition of the stream bacterial communities. RESULTS: Random forest modelling allowed us to use bacterial community composition to predict upstream catchment land use with 65% accuracy; urban sites were correctly assigned 90% of the time. Despite the variation inherent when sampling across a ~ 1000-km distance, bacterial community data could correctly differentiate undisturbed sites, grouped by their dominant environmental properties, with 75% accuracy. The positive correlations between actual values and those predicted by the models built using the stream biofilm bacterial data ranged from weak (average log N concentration in the stream water, R2 = 0.02) to strong (annual mean air temperature, R2 = 0.69). CONCLUSIONS: Freshwater bacterial community data provide useful insights into land use impacts on stream ecosystems; they may be used as an additional measure to screen stream catchment attributes.

Soil conditions are a more important determinant of microbial community composition and functional potential than neighboring plant diversity.

Replanting is an important tool for ecological recovery. Management strategies, such as planting areas with monocultures or species mixtures, have implications for restoration success. We used 16S and ITS rRNA gene amplicon sequencing and shotgun metagenomics to assess how the diversity of neighboring tree species impacted soil bacterial and fungal communities, and their functional potential, within the root zone of manuka (Leptospermum scoparium) trees. We compared data from monoculture and mixed tree species plots and confirmed that soil microbial taxonomic and functional community profiles significantly differed (p < 0.001). Compared to the diversity of neighboring tree species within the plot, soil environmental conditions and geographic distance was more important for structuring the microbial communities. The bacterial communities appeared more impacted by soil conditions, while the fungal communities displayed stronger spatial structuring, possibly due to wider bacterial dispersal. The different mechanisms structuring bacterial and fungal communities could have implications for ecological restoration outcomes.

Uncoupled: investigating the lack of correlation between the transcription of putative plastic-degrading genes in the global ocean microbiome and marine plastic pollution.

BACKGROUND: Plastic pollution is a severe threat to marine ecosystems. While some microbial enzymes can degrade certain plastics, the ability of the global ocean microbiome to break down diverse environmental plastics remains limited. We employed metatranscriptomic data from an international ocean survey to explore global and regional patterns in microbial plastic degradation potential. RESULTS: On a global oceanic scale, we found no significant correlation between levels of plastic pollution and the expression of genes encoding enzymes putatively identified as capable of plastic degradation. Even when looking at different regional scales, ocean depth layers, or plastic types, we found no strong or even moderate correlation between plastic pollution and relative abundances of transcripts for enzymes with presumed plastic biodegradation potential. Our data, however, indicate that microorganisms in the Southern Ocean show a higher potential for plastic degradation, making them more appealing candidates for bioprospecting novel plastic-degrading enzymes. CONCLUSION: Our research contributes to understanding the complex global relationship between plastic pollution and microbial plastic degradation potential. We reveal that the transcription of putative plastic-degrading genes in the global ocean microbiome does not correlate to marine plastic pollution, highlighting the ongoing danger that plastic poses to marine environments threatened by plastic pollution.

Characterization of the transcriptional effects of the plastic additive dibutyl phthalate alone and in combination with microplastic on the green-lipped mussel Perna canaliculus.

The presence and persistence of microplastics (MPs) in diverse aquatic environments are of global concern. Microplastics can impact marine organisms via direct physical interaction and the release of potentially harmful chemical additives incorporated into the plastic. These chemicals are physically bound to the plastic matrix and can leach out. The hazards associated with chemical additives to exposed organisms is not well characterized. We investigated the hazards of plastic additives leaching from plastic. We used the common plasticizer dibutyl phthalate (DBP) as a chemical additive proxy and the New Zealand green-lipped mussel (Perna canaliculus) as a model. We used early-adult P. canaliculus exposed to combinations of virgin and DBP-spiked polyvinyl chloride (PVC), MPs, and DBP alone for 7 days. Whole transcriptome sequencing (RNA-seq) was conducted to assess whether leaching of DBP from MPs poses a hazard. The differences between groups were evaluated using pairwise permutational multivariate analysis of variance (PERMANOVA), and all treatments were significantly different from controls. In addition, a significant difference was seen between DBP and PVC MP treatment. Transcriptome analysis revealed that mussels exposed to DBP alone had the most differentially expressed genes (914), followed by PVC MP + DBP (448), and PVC MP (250). Gene ontology functional analysis revealed that the most enriched pathway types were in cellular metabolism, immune response, and endocrine disruption. Microplastic treatments enriched numerous pathways related to cellular metabolism and immune response. The combined exposure of PVC MP + DBP appears to cause combined effects, suggesting that DBP is bioavailable to the exposed mussels in the PVC MP + DBP treatment. Our results support the hypothesis that chemical additives are potentially an important driver of MP toxicity. Environ Toxicol Chem 2024;43:1604-1614. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

An investigation into the stability and degradation of plastics in aquatic environments using a large-scale field-deployment study.

The fragmentation of plastic debris is a key pathway to the formation of microplastic pollution. These disintegration processes depend on the materials' physical and chemical characteristics, but insight into these interrelationships is still limited, especially under natural conditions. Five plastics of known polymer/additive compositions and processing histories were deployed in aquatic environments and recovered after six and twelve months. The polymer types used were linear low density polyethylene (LLDPE), oxo-degradable LLDPE (oxoLLDPE), poly(ethylene terephthalate) (PET), polyamide-6 (PA6), and poly(lactic acid) (PLA). Four geographically distinct locations across Aotearoa/New Zealand were chosen: three marine sites and a wastewater treatment plant (WWTP). Accelerated UV-weathering under controlled laboratory conditions was also carried out to evaluate artificial ageing as a model for plastic degradation in the natural environment. The samples' physical characteristics and surface microstructures were studied for each deployment location and exposure time. The strongest effects were found for oxoLLDPE upon artificial ageing, with increased crystallinity, intense surface cracking, and substantial deterioration of its mechanical properties. However, no changes to the same extent were found after recovery of the deployed material. In the deployment environments, the chemical nature of the plastics was the most relevant factor determining their behaviours. Few significant differences between the four aquatic locations were identified, except for PA6, where indications for biological surface degradation were found only in seawater, not the WWTP. In some cases, artificial ageing reasonably mimicked the changes which some plastic properties underwent in aquatic environments, but generally, it was no reliable model for natural degradation processes. The findings from this study have implications for the understanding of the initial phases of plastic degradation in aquatic environments, eventually leading to microplastics formation. They can also guide the interpretation of accelerated laboratory ageing for the fate of aquatic plastic pollution, and for the testing of aged plastic samples.

Leaching and transformation of chemical additives from weathered plastic deployed in the marine environment.

Plastic pollution causes detrimental environmental impacts, which are increasingly attributed to chemical additives. However, the behaviour of plastic additives in the marine environment is poorly understood. We used a marine deployment experiment to examine the impact of weathering on the extractables profile, analysed by liquid chromatography-mass spectrometry, of four plastics at two locations over nine months in Aotearoa/New Zealand. The concentration of additives in polyethylene and oxo-degradable polyethylene were strongly influenced by artificial weathering, with deployment location and time less influential. By comparison, polyamide 6 and polyethylene terephthalate were comparatively inert with minimal change in response to artificial weathering or deployment time. Non-target analysis revealed extensive differentiation between non-aged and aged polyethylene after deployment, concordant with the targeted analysis. These observations highlight the need to consider the impact of leaching and weathering on plastic composition when quantifying the potential impact and risk of plastic pollution within receiving environments.

Soil bacterial community composition is more stable in kiwifruit orchards relative to phyllosphere communities over time.

BACKGROUND: Soil and phyllosphere (leaves and fruit) microbes play critical roles in the productivity and health of crops. However, microbial community dynamics are currently understudied in orchards, with a limited number incorporating temporal monitoring. We used 16S rRNA gene amplicon sequencing to investigate bacterial community temporal dynamics and community assembly processes on the leaves and fruit, and in the soil of 12 kiwifruit orchards across a cropping season in New Zealand. RESULTS: Community composition significantly differed (P < 0.001) among the three sample types. However, the communities in the phyllosphere substrates more closely resembled each other, relative to the communities in the soil. There was more temporal stability in the soil bacterial community composition, relative to the communities residing on the leaves and fruit, and low similarity between the belowground and aboveground communities. Bacteria in the soil were more influenced by deterministic processes, while stochastic processes were more important for community assembly in the phyllosphere. CONCLUSIONS: The higher temporal variability and the stochastic nature of the community assembly processes observed in the phyllosphere communities highlights why predicting the responsiveness of phyllosphere communities to environmental change, or the likelihood of pathogen invasion, can be challenging. The relative temporal stability and the influence of deterministic selection on soil microbial communities suggests a greater potential for their prediction and reliable manipulation.

A spatio-temporal analysis of marine diatom communities associated with pristine and aged plastics.

Complex microbial communities colonize plastic substrates over time, strongly influencing their fate and potential impacts on marine ecosystems. Among the first colonizers, diatoms play an important role in the development of this 'plastiphere'. We investigated 936 biofouling samples and the factors influencing diatom communities associated with plastic colonization. These factors included geographic location (up to 800 km apart), duration of substrate submersion (1 to 52 weeks), plastics (5 polymer types) and impact of artificial ageing with UV light. Diatom communities colonizing plastic debris were primarily determined by their geographic location and submersion time, with the strongest changes occurring within two weeks of submersion. Several taxa were identified as early colonizers (e.g. Cylindrotheca, Navicula and Nitzschia spp.) with known strong adhesion capabilities. To a lesser extent, plastic-type and UV-ageing significantly affected community composition, with 14 taxa showing substrate-specificity. This study highlights the role of plastics types-state for colonization in the ocean.

Comparison between droplet digital PCR and reverse transcription-quantitative PCR methods to measure ecotoxicology biomarkers.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is currently the gold-standard technique for detecting and quantifying messenger RNA. However, without proper validation, the method may produce artefactual and non-reproducible cycle threshold values generating poor-quality data. The newer droplet digital PCR (ddPCR) method allows for the absolute quantification of targeted nucleic acids providing more sensitive and accurate measurements without requiring external standards. This study compared these two PCR-based methods to measure the expression of well-documented genes used in ecotoxicology studies. We exposed Mediterranean mussels (Mytilus galloprovincialis) to copper and analyzed gene expression in gills and digestive glands using RT-qPCR and ddPCR assays. A step-by-step methodology to optimize and compare the two technologies is described. After ten-fold serial complementary DNA dilution, both RT-qPCR and ddPCR exhibited comparable linearity and efficiency and produced statistically similar results. We conclude that ddPCR is a suitable method to assess gene expression in an ecotoxicological context. However, RT-qPCR has a shorter processing time and remains more cost-effective.

The soil microbiome: An essential, but neglected, component of regenerative agroecosystems.

Regenerative agriculture (RA) is gaining traction globally as an approach for meeting growing food demands while avoiding, or even remediating, the detrimental environmental consequences associated with conventional farming. Momentum is building for science to provide evidence for, or against, the putative ecosystem benefits of RA practices relative to conventional farming. In this perspective article, we advance the argument that consideration of the soil microbiome in RA research is crucial for disentangling the varied and complex relationships RA practices have with the biotic and abiotic environment, outline the expected changes in soil microbiomes under RA, and make recommendations for designing research that will answer the outstanding questions on the soil microbiome under RA. Ultimately, deeper insights into the role of microbial communities in RA soils will allow the development of biologically relevant monitoring tools which will support land managers in addressing the key environmental issues associated with agriculture.

Does plastic type matter? Insights into non-indigenous marine larvae recruitment under controlled conditions.

Marine plastic debris (MPD) are a global threat to marine ecosystems. Among countless ecosystem impacts, MPD can serve as a vector for marine 'hitchhikers' by facilitating transport and subsequent spread of unwanted pests and pathogens. The transport and spread of these non-indigenous species (NIS) can have substantial impacts on native biodiversity, ecosystem services/functions and hence, important economic consequences. Over the past decade, increasing research interest has been directed towards the characterization of biological communities colonizing plastic debris, the so called Plastisphere. Despite remarkable advances in this field, little is known regarding the recruitment patterns of NIS larvae and propagules on MPD, and the factors influencing these patterns. To address this knowledge gap, we used custom-made bioassay chambers and ran four consecutive bioassays to compare the settlement patterns of four distinct model biofouling organisms' larvae, including the three notorious invaders Crassostrea gigas, Ciona savignyi and Mytilus galloprovincialis, along with one sessile macro-invertebrate Spirobranchus cariniferus, on three different types of polymers, namely Low-Linear Density Polyethylene (LLDPE), Polylactic Acid (PLA), Nylon-6, and a glass control. Control bioassay chambers were included to investigate the microbial community composition colonizing the different substrates using 16S rRNA metabarcoding. We observed species-specific settlement patterns, with larvae aggregating on different locations on the substrates. Furthermore, our results revealed that C. savignyi and S. cariniferus generally favoured Nylon and PLA, whereas no specific preferences were observed for C. gigas and M. galloprovincialis. We did not detect significant differences in bacterial community composition between the tested substrates. Taken together, our results highlight the complexity of interactions between NIS larvae and plastic polymers. We conclude that several factors and their potential interactions influenced the results of this investigation, including: (i) species-specific larval biological traits and ecology; (ii) physical and chemical composition of the substrates; and (iii) biological cues emitted by bacterial biofilm and the level of chemosensitivity of the different NIS larvae. To mitigate the biosecurity risks associated with drifting plastic debris, additional research effort is critical to effectively decipher the mechanisms involved in the recruitment of NIS on MPD.

Influence of management practice on the microbiota of a critically endangered species: a longitudinal study of kakapo chick faeces and associated nest litter.

BACKGROUND: The critically endangered kakapo is a flightless, nocturnal parrot endemic to Aotearoa New Zealand. Recent efforts to describe the gastrointestinal microbial community of this threatened herbivore revealed a low-diversity microbiota that is often dominated by Escherichia-Shigella bacteria. Given the importance of associated microbial communities to animal health, and increasing appreciation of their potential relevance to threatened species conservation, we sought to better understand the development of this unusual gut microbiota profile. To this end, we conducted a longitudinal analysis of faecal material collected from kakapo chicks during the 2019 breeding season, in addition to associated nest litter material. RESULTS: Using an experimental approach rarely seen in studies of threatened species microbiota, we evaluated the impact of a regular conservation practice on the developing kakapo microbiota, namely the removal of faecal material from nests. Artificially removing chick faeces from nests had negligible impact on bacterial community diversity for either chicks or nests (p > 0.05). However, the gut microbiota did change significantly over time as chick age increased (p < 0.01), with an increasing relative abundance of Escherichia-Shigella coli over the study period and similar observations for the associated nest litter microbiota (p < 0.01). Supplementary feeding substantially altered gut bacterial diversity of kakapo chicks (p < 0.01), characterised by a significant increase in Lactobacillus bacteria. CONCLUSIONS: Overall, chick age and hand rearing conditions had the most marked impact on faecal bacterial communities. Similarly, the surrounding nest litter microbiota changed significantly over time since a kakapo chick was first placed in the nest, though we found no evidence that removal of faecal material influenced the bacterial communities of either litter or faecal samples. Taken together, these observations will inform ongoing conservation and management of this most enigmatic of bird species.

Time after time: detecting annual patterns in stream bacterial biofilm communities.

To quantify the major environmental drivers of stream bacterial population dynamics, we modelled temporal differences in stream bacterial communities to quantify community shifts, including those relating to cyclical seasonal variation and more sporadic bloom events. We applied Illumina MiSeq 16S rRNA bacterial gene sequencing of 892 stream biofilm samples, collected monthly for 36-months from six streams. The streams were located a maximum of 118 km apart and drained three different catchment types (forest, urban and rural land uses). We identified repeatable seasonal patterns among bacterial taxa, allowing their separation into three ecological groupings, those following linear, bloom/trough and repeated, seasonal trends. Various physicochemical parameters (light, water and air temperature, pH, dissolved oxygen, nutrients) were linked to temporal community changes. Our models indicate that bloom events and seasonal episodes modify biofilm bacterial populations, suggesting that distinct microbial taxa thrive during these events including non-cyanobacterial community members. These models could aid in determining how temporal environmental changes affect community assembly and guide the selection of appropriate statistical models to capture future community responses to environmental change.

PlasticDB: a database of microorganisms and proteins linked to plastic biodegradation.

The number of publications reporting putative plastic-degrading microbes and proteins is continuously increasing, necessitating the compilation of these data and the development of tools to facilitate their analysis. We developed the PlasticDB web application to address this need, which comprises a database of microorganisms and proteins reported to biodegrade plastics. Associated metadata, such as the techniques utilized to assess biodegradation, the environmental source of microbial isolate and presumed thermophilic traits are also reported. Proteins in the database are categorized according to the plastic type they are reported to degrade. Each protein structure has been predicted in silico and can be visualized or downloaded for further investigation. In addition to standard database functionalities, such as searching, filtering and retrieving database records, we implemented several analytical tools that accept inputs, including gene, genome, metagenome, transcriptomes, metatranscriptomes and taxa table data. Users can now analyze their datasets for the presence of putative plastic-degrading species and potential plastic-degrading proteins and pathways from those species. Database URL:http://plasticdb.org.

Assessing integrated biomarkers of triplefin fish Forsterygion capito inhabiting contaminated marine water - A multivariate approach.

The presence of multiple chemicals in aquatic ecosystems makes evaluation of their real impact on the biota difficult. Integrated biomarkers are therefore needed to evaluate how these chemicals contribute to environmental degradation. The aims of the present study were to evaluate responses to and effects of marine pollution using a series of biomarkers through multivariate analyses. Transcriptional responses of cyp1a (cytochrome P450), mt (metallothionein), vtg (vitellogenin) and cyp19b (cytochrome P450 aromatase); branchial and hepatic histological alterations; and Fulton condition factors (CF) were evaluated, as well as the metals and polycyclic aromatic hydrocarbons present in Forsterygion capito in Auckland, New Zealand. Sites were selected along a contamination gradient: four highly contaminated sites and four less contaminated. Molecular responses with a higher relative expression of the mt and cyp1a genes were detected at a highly contaminated site (Panmure). Several histological lesion types were found in the livers of fish inhabiting both types of sites, but gill lesions were present primarily at highly contaminated sites. In terms of general health status, the lowest CF values were overwhelmingly found in fish from the same site (Panmure). The multivariate approach revealed that telangiectasia and hyperplasia were associated with the presence of chemicals, and these showed negative associations with the CF values, with fish from three highly contaminated sites being most affected. In conclusion, the multivariate approach helped to integrate these biological markers in this blennioid fish, thus providing a more holistic view of the complex chemical mixtures involved. Future studies should implement these analyses.

Genome Streamlining, Plasticity, and Metabolic Versatility Distinguish Co-occurring Toxic and Nontoxic Cyanobacterial Strains of Microcoleus.

Harmful cyanobacterial bloom occurrences have increased worldwide due to climate change and eutrophication, causing nuisance and animal deaths. Species from the benthic cyanobacterial genus Microcoleus are ubiquitous and form thick mats in freshwater systems, such as rivers, that are sometimes toxic due to the production of potent neurotoxins (anatoxins). Anatoxin-producing (toxic) strains typically coexist with non-anatoxin-producing (nontoxic) strains in mats, although the reason for this is unclear. To determine the genetic mechanisms differentiating toxic and nontoxic Microcoleus, we sequenced and assembled genomes from 11 cultures and compared these to another 31 Microcoleus genomes. Average nucleotide identities (ANI) indicate that toxic and nontoxic strains are distinct species (ANI, <95%), and only 6% of genes are shared across all 42 genomes, suggesting a high level of genetic divergence among Microcoleus strains. Comparative genomics showed substantial genome streamlining in toxic strains and a potential dependency on external sources for thiamine and sucrose. Toxic and nontoxic strains are further differentiated by an additional set of putative nitrate transporter (nitrogen uptake) and cyanophycin (carbon and nitrogen storage) genes, respectively. These genes likely confer distinct competitive advantages based on nutrient availability and suggest nontoxic strains are more robust to nutrient fluctuations. Nontoxic strains also possess twice as many transposable elements, potentially facilitating greater genetic adaptation to environmental changes. Our results offer insights into the divergent evolution of Microcoleus strains and the potential for cooperative and competitive interactions that contribute to the co-occurrence of toxic and nontoxic species within mats. IMPORTANCE Microcoleus autumnalis, and closely related Microcoleus species, compose a geographically widespread group of freshwater benthic cyanobacteria. Canine deaths due to anatoxin-a poisoning, following exposure to toxic proliferations, have been reported globally. While Microcoleus proliferations are on the rise, the mechanisms underpinning competition between, or coexistence of, toxic and nontoxic strains are unknown. This study identifies substantial genetic differences between anatoxin-producing and non-anatoxin-producing strains, pointing to reduced metabolic flexibility in toxic strains, and potential dependence on cohabiting nontoxic strains. Results provide insights into the metabolic and evolutionary differences between toxic and nontoxic Microcoleus, which may assist in predicting and managing aquatic proliferations.

Microbial river-to-sea continuum: gradients in benthic and planktonic diversity, osmoregulation and nutrient cycling.

BACKGROUND: Coastal aquatic ecosystems include chemically distinct, but highly interconnected environments. Across a freshwater-to-marine transect, aquatic communities are exposed to large variations in salinity and nutrient availability as tidal cycles create periodic fluctuations in local conditions. These factors are predicted to strongly influence the resident microbial community structure and functioning, and alter the structure of aquatic food webs and biogeochemical cycles. Nevertheless, little is known about the spatial distribution of metabolic properties across salinity gradients, and no study has simultaneously surveyed the sediment and water environments. Here, we determined patterns and drivers of benthic and planktonic prokaryotic and microeukaryotic community assembly across a river and tidal lagoon system by collecting sediments and planktonic biomass at nine shallow subtidal sites in the summer. Genomic and transcriptomic analyses, alongside a suite of complementary geochemical data, were used to determine patterns in the distribution of taxa, mechanisms of salt tolerance, and nutrient cycling. RESULTS: Taxonomic and metabolic profiles related to salt tolerance and nutrient cycling of the aquatic microbiome were found to decrease in similarity with increasing salinity, and distinct trends in diversity were observed between the water column and sediment. Non-saline and saline communities adopted divergent strategies for osmoregulation, with an increase in osmoregulation-related transcript expression as salinity increased in the water column due to lineage-specific adaptations to salt tolerance. Results indicated a transition from phosphate limitation in freshwater habitats to nutrient-rich conditions in the brackish zone, where distinct carbon, nitrogen and sulfur cycling processes dominated. Phosphorus acquisition-related activity was highest in the freshwater zone, along with dissimilatory nitrate reduction to ammonium in freshwater sediment. Activity associated with denitrification, sulfur metabolism and photosynthesis were instead highest in the brackish zone, where photosynthesis was dominated by distinct microeukaryotes in water (Cryptophyta) and sediment (diatoms). Despite microeukaryotes and archaea being rare relative to bacteria, results indicate that they contributed more to photosynthesis and ammonia oxidation, respectively. CONCLUSIONS: Our study demonstrates clear freshwater-saline and sediment-water ecosystem boundaries in an interconnected coastal aquatic system and provides a framework for understanding the relative importance of salinity, planktonic-versus-benthic habitats and nutrient availability in shaping aquatic microbial metabolic processes, particularly in tidal lagoon systems. Video abstract.

Phospholipid fatty acid (PLFA) analysis as a tool to estimate absolute abundances from compositional 16S rRNA bacterial metabarcoding data.

Microbial biodiversity monitoring through the analysis of DNA extracted from environmental samples is increasingly popular because it is perceived as being rapid, cost-effective, and flexible concerning the sample types studied. DNA can be extracted from diverse media before high-throughput sequencing of the prokaryotic 16S rRNA gene is used to characterize the taxonomic diversity and composition of the sample (known as metabarcoding). While sources of bias in metabarcoding methodologies are widely acknowledged, previous studies have focused mainly on the effects of these biases within a single substrate type, and relatively little is known of how these vary across substrates. We investigated the effect of substrate type (water, microbial mats, lake sediments, stream sediments, soil and a mock microbial community) on the relative performance of DNA metabarcoding in parallel with phospholipid fatty acid (PLFA) analysis. Quantitative estimates of the biomass of different taxonomic groups in samples were made through the analysis of PLFAs, and these were compared to the relative abundances of microbial taxa estimated from metabarcoding. Furthermore, we used the PLFA-based quantitative estimates of the biomass to adjust relative abundances of microbial groups determined by metabarcoding to provide insight into how the biomass of microbial taxa from PLFA analysis can improve understanding of microbial communities from environmental DNA samples. We used two sets of PLFA biomarkers that differed in their number of PLFAs to evaluate how PLFA biomarker selection influences biomass estimates. Metabarcoding and PLFA analysis provided significantly different views of bacterial composition, and these differences varied among substrates. We observed the most notable differences for the Gram-negative bacteria, which were overrepresented by metabarcoding in comparison to PLFA analysis. In contrast, the relative biomass and relative sequence abundances aligned reasonably well for Cyanobacteria across the tested freshwater substrates. Adjusting relative abundances of microbial taxa estimated by metabarcoding with PLFA-based quantification estimates of the microbial biomass led to significant changes in the microbial community compositions in all substrates. We recommend including independent estimates of the biomass of microbial groups to increase comparability among metabarcoding libraries from environmental samples, especially when comparing communities associated with different substrates.

Measuring change in biological communities: multivariate analysis approaches for temporal datasets with low sample size.

Effective and robust ways to describe, quantify, analyse, and test for change in the structure of biological communities over time are essential if ecological research is to contribute substantively towards understanding and managing responses to ongoing environmental changes. Structural changes reflect population dynamics, changes in biomass and relative abundances of taxa, and colonisation and extinction events observed in samples collected through time. Most previous studies of temporal changes in the multivariate datasets that characterise biological communities are based on short time series that are not amenable to data-hungry methods such as multivariate generalised linear models. Here, we present a roadmap for the analysis of temporal change in short-time-series, multivariate, ecological datasets. We discuss appropriate methods and important considerations for using them such as sample size, assumptions, and statistical power. We illustrate these methods with four case-studies analysed using the R data analysis environment.

Changes in the analysis of temporal community dynamics data: a 29-year literature review.

BACKGROUND: Understanding how biological communities change over time is of increasing importance as Earth moves into the Anthropocene. A wide variety of methods are used for multivariate community analysis and are variously applied to research that aims to characterise temporal dynamics in community composition. Understanding these methods and how they are applied is useful for determining best practice in community ecology. METHODOLOGY: We reviewed the ecological literature from 1990 to 2018 that used multivariate methods to address questions of temporal community dynamics. For each paper that fulfilled our search criteria, we recorded the types of multivariate analysis used to characterise temporal community dynamics in addition to the research aim, habitat type, location, taxon and the experimental design. RESULTS: Most studies had relatively few temporal replicates; the median number was seven time points. Nearly 70% of studies applied more than one analysis method; descriptive methods such as bar graphs and ordination were the most commonly applied methods. Surprisingly, the types of analyses used were only related to the number of temporal replicates, but not to research aim or any other aspects of experimental design such as taxon, or habitat or year of study. CONCLUSIONS: This review reveals that most studies interested in understanding community dynamics use relatively short time series meaning that several, more sophisticated, temporal analyses are not widely applicable. However, newer methods using multivariate dissimilarities are growing in popularity and many can be applied to time series of any length.