Microbial Community Structure in a Malaysian Tropical Peat Swamp Forest: The Influence of Tree Species and Depth

Tropical peat swamp forests sequester globally significant stores of carbon in deep layers of waterlogged, anoxic, acidic and nutrient-depleted peat. The roles of microbes in supporting these forests through the formation of peat, carbon sequestration and nutrient cycling are virtually unknown. This study investigated physicochemical peat properties and microbial diversity between three dominant tree species: Shorea uliginosa (Dipterocarpaceae), Koompassia malaccensis (legumes associated with nitrogen-fixing bacteria), Eleiodoxa conferta (palm) and depths (surface, 45 and 90 cm) using microbial 16S rRNA gene amplicon sequencing. Water pH, oxygen, nitrogen, phosphorus, total phenolic contents and C/N ratio differed significantly between depths, but not tree species. Depth also strongly influenced microbial diversity and composition, while both depth and tree species exhibited significant impact on the archaeal communities. Microbial diversity was highest at the surface, where fresh leaf litter accumulates, and nutrient supply is guaranteed. Nitrogen was the core parameter correlating to microbial communities, but the interactive effects from various environmental variables displayed significant correlation to relative abundance of major microbial groups. Proteobacteria was the dominant phylum and the most abundant genus, Rhodoplanes, might be involved in nitrogen fixation. The most abundant methanogens and methanotrophs affiliated, respectively, to families Methanomassiliicoccaceae and Methylocystaceae. Our results demonstrated diverse microbial communities and provide valuable insights on microbial ecology in these extreme ecosystems

Hochdurchsatzerfassung von Biodiversität - Stärken und Grenzen von Meta-barcoding

Traditional species identification based on morphological characters is laborious and requires expert knowledge. It is further complicated in the case of species assemblages or degraded and processed material. DNA-barcoding, species identification based on genetic data, has become a suitable alternative, yet species assemblages are still difficult to study. In the past decade meta-barcoding has widely been adopted for the study of species communities, due to technological advances in modern sequencing platforms and because manual separation of individual specimen is not required. Here, meta-barcoding is put into context and applied to the study of bee-collected pollen as well as bacterial communities. These studies provide the basis for a critical evaluation of the powers and limitations of meta-barcoding. Advantages identified include species identification without the need for expert knowledge as well as the high throughput of samples and sequences. In microbiology, meta-barcoding can facilitate directed cultivation of taxa of interest identified with meta-barcoding data. Disadvantages include insufficient species resolution due to short read lengths and incomplete reference databases, as well as limitations in abundance estimation of taxa and functional profiling. Despite these, meta-barcoding is a powerful method for the analysis of species communities and holds high potential especially for automated biomonitoring.Traditionelle Methoden der Identifizierung von Organismen anhand von morphologischen Merkmalen sind arbeits- und zeitaufwendig und benötigen Expertenkenntnisse der Morphologie. Weitere Probleme liegen in der Analyse von Artgemeinschaften und prozessiertem Material. DNA-barcoding, Artbestimmung anhand von genetischen Merkmalen, hat sich als Alternative herausgebildet, jedoch sind Artgemeinschaften nach wie vor schwierig zu analysieren. Im vergangenen Jahrzehnt wurde meta-barcoding zur Analyse von Artgemeinschaften entwickelt; insbesondere durch die Weiterentwicklung moderner Sequenziergeräte und da eine Auftrennung der Organismen innerhalb einer Gemeinschaft nicht mehr notwendig ist. In der vorliegenden Arbeit wurde zunächst ein Überblick über meta-barcoding erstellt. Die Methode wurde dann für die Analyse von Bienen-gesammeltem Pollen und Bakteriengemeinschaften angewandt. Diese Studien bilden eine gute Basis, um die Vor- und Nachteile von meta-barcoding kritisch zu bewerten. Vorteile beinhalten unter anderem, dass Organismen bestimmt werden können, ohne dass Expertenkenntnisse notwendig sind, sowie der hohe Durchsatz von Proben und Sequenzen. In der Mikrobiologie kann meta-barcoding eine gerichtete Kultivierung von Bakterien erleichtern, die durch meta-barcoding als Zielorganismen indentifiziert wurden. Nachteile finden sich in der manchmal noch unzureichenden Unterscheidung nah ver- wandter Arten aufgrund von kurzen Sequenzlängen und lückenhaften Referenzdatenbanken, sowie Einschränkungen in der Abschätzung von Abundanzen und Funktionen der Organismen innerhalb der Artgemeinschaft. Trotz dieser Problematiken ist meta-barcoding eine leistungsstarke Methode für die Analyse von Artgemeinschaften und ist besonders vielversprechend für automatisiertes Bio-Monitoring

BeeeDNA-ReferenceDatabases

<p>Reference databases used for taxonomic classification of sequence data belonging to the publication<strong> </strong><i>'</i>BEE-quest of the nest: A novel method for eDNA-based, non-lethal detection of cavity-nesting hymenopterans and other arthropods<i>'</i> and GitHub repository: https://github.com/BWobbii/BeeeDNA</p><p> </p&gt

Supplementary material 2 from: Sickel W, Zizka V, Scherges A, Bourlat SJ, Dieker P (2023) Abundance estimation with DNA metabarcoding – recent advancements for terrestrial arthropods. Metabarcoding and Metagenomics 7: e112290. https://doi.org/10.3897/mbmg.7.112290

Literature collectio

Increased efficiency in identifying mixed pollen samples by meta-barcoding with a dual-indexing approach

Background Meta-barcoding of mixed pollen samples constitutes a suitable alternative to conventional pollen identification via light microscopy. Current approaches however have limitations in practicability due to low sample throughput and/or inefficient processing methods, e.g. separate steps for amplification and sample indexing. Results We thus developed a new primer-adapter design for high throughput sequencing with the Illumina technology that remedies these issues. It uses a dual-indexing strategy, where sample-specific combinations of forward and reverse identifiers attached to the barcode marker allow high sample throughput with a single sequencing run. It does not require further adapter ligation steps after amplification. We applied this protocol to 384 pollen samples collected by solitary bees and sequenced all samples together on a single Illumina MiSeq v2 flow cell. According to rarefaction curves, 2,000–3,000 high quality reads per sample were sufficient to assess the complete diversity of 95% of the samples. We were able to detect 650 different plant taxa in total, of which 95% were classified at the species level. Together with the laboratory protocol, we also present an update of the reference database used by the classifier software, which increases the total number of covered global plant species included in the database from 37,403 to 72,325 (93% increase). Conclusions This study thus offers improvements for the laboratory and bioinformatical workflow to existing approaches regarding data quantity and quality as well as processing effort and cost-effectiveness. Although only tested for pollen samples, it is furthermore applicable to other research questions requiring plant identification in mixed and challenging samples

Abundance estimation with DNA metabarcoding – recent advancements for terrestrial arthropods

Biodiversity is declining at alarming rates worldwide and large-scale monitoring is urgently needed to understand changes and their drivers. While classical taxonomic identification of species is time and labor intensive, the combination with DNA-based methods could upscale monitoring activities to achieve larger spatial coverage and increased sampling effort. However, challenges remain for DNA-based methods when species counts and/or biomass estimates are required. Several methodological advancements exist to improve the potential of DNA metabarcoding for abundance analysis, which however need further evaluation. Here, we discuss laboratory, as well as some bioinformatic adjustments to DNA metabarcoding workflows regarding their potential to achieve species abundance estimation from arthropod community samples. Our review includes pre-laboratory processing methods such as specimen photography, laboratory methods such as the use of spike-in DNA as an internal standard and bioinformatic advancements like correction factors. We conclude that specimen photography coupled with DNA metabarcoding currently promises the greatest potential to achieve species counts and biomass estimates, but that approaches such as spike-ins and correction factors are promising methods to pursue further

Supplementary material 4 from: Sickel W, Zizka V, Scherges A, Bourlat SJ, Dieker P (2023) Abundance estimation with DNA metabarcoding – recent advancements for terrestrial arthropods. Metabarcoding and Metagenomics 7: e112290. https://doi.org/10.3897/mbmg.7.112290

Evaluation of methodological approache

Abundance estimation with DNA metabarcoding – recent advancements for terrestrial arthropods

Biodiversity is declining at alarming rates worldwide and large-scale monitoring is urgently needed to understand changes and their drivers. While classical taxonomic identification of species is time and labour intensive, the combination with DNA-based methods could upscale monitoring activities to achieve larger spatial coverage and increased sampling effort. However, challenges remain for DNA-based methods when the number of individuals per species and/or biomass estimates are required. Several methodological advancements exist to improve the potential of DNA metabarcoding for abundance analysis, which however need further evaluation. Here, we discuss laboratory, as well as some bioinformatic adjustments to DNA metabarcoding workflows regarding their potential to achieve species abundance estimation from arthropod community samples. Our review includes pre-laboratory processing methods such as specimen photography, laboratory methods such as the use of spike-in DNA as an internal standard and bioinformatic advancements like correction factors. We conclude that specimen photography coupled with DNA metabarcoding currently promises the greatest potential to achieve estimates of the number of individuals per species and biomass estimates, but that approaches such as spike-ins and correction factors are promising methods to pursue further

Supplementary material 1 from: Sickel W, Zizka V, Scherges A, Bourlat SJ, Dieker P (2023) Abundance estimation with DNA metabarcoding – recent advancements for terrestrial arthropods. Metabarcoding and Metagenomics 7: e112290. https://doi.org/10.3897/mbmg.7.112290

Background informatio

Supplementary material 3 from: Sickel W, Zizka V, Scherges A, Bourlat SJ, Dieker P (2023) Abundance estimation with DNA metabarcoding – recent advancements for terrestrial arthropods. Metabarcoding and Metagenomics 7: e112290. https://doi.org/10.3897/mbmg.7.112290

Categories assessed in the literature revie