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Alpha-diversity indices are an essential tool for describing and comparing biodiversity. Microbial ecologists apply indices originally intended for, or adopted by, macroecology to address questions relating to taxonomy (conserved marker) and function (metagenome-based data). In this Perspective piece, I begin by discussing the nature and mathematical quirks important for interpreting routinely employed alpha-diversity indices. Secondly, I propose a metagenomic alpha-diversity index (MD) that measures the (dis)similarity of protein-encoding genes within a community. MD has defined limits, whereby a community comprised mostly of similar, poorly diverse protein-encoding genes pulls the index to the lower limit, while a community rich in divergent homologs and unique genes drives it toward the upper limit. With data acquired from an in silico and three in situ metagenome studies, I derive MD and typical alpha-diversity indices applied to taxonomic (ribosomal rRNA) and functional (all protein-encoding) genes, and discuss their relationships with each other. Not all alpha-diversity indices detect biological trends, and taxonomic does not necessarily follow functional biodiversity. Throughout, I explain that protein Richness and MD provide complementary and easily interpreted information, while probability-based indices do not. Finally, considerations regarding the unique nature of microbial metagenomic data and its relevance for describing functional biodiversity are discussed., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

Despite adoption of high-throughput sequencing of PCR-amplified microbial taxonomic markers for ecological analyses, distinct approaches for preparing amplicon libraries exist. One approach utilises long fusion primers and a single PCR (one-step) while another utilises shorter primers in a first reaction, before transferring diluted amplicons to a second reaction for barcode index incorporation (two-step). We investigated whether transferring diluted amplicons risked creating artificially simplified, poorly diverse communities. In soils from three sites with paired cropland and forest, one-step yielded higher alpha-diversity indices, including detection of two-four times more unique taxa. Modelling expected taxa per sequence observation predicted that one-step reaches full coverage by 104 sequences per sample while two-step needs 105-109. Comparisons of rank abundance demonstrated that two-step covered only 38%-69% of distributions. Beta-diversity showed better separation of communities in response to land use change under one-step, although both approaches showed a significant effect. Driving differences was underestimation of relatively minor taxa with the two-step procedure. These taxa were low in abundance, yet play important roles in carbon cycling, secondary metabolite production, anaerobic metabolism, and bacterial predation. We conclude that one-step amplicon libraries are advisable for studies focussed on diversity or relatively minor yet functionally important taxa., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

Crop harvest followed by a fallow period can act as a disturbance on soil microbial communities. Cropping systems intended to improve alpha-diversity of communities may also confer increased compositional stability during succeeding growing seasons. Over a single growing season in a long-term (18 year) agricultural field experiment incorporating conventional (CON), conservation (CA), organic (ORG) and integrated (INT) cropping systems, temporal changes in prokaryote, fungal and arbuscular mycorrhizal fungi (AMF) communities were investigated overwinter, during crop growth and at harvest. While certain prokaryote phyla were influenced by cropping system (e.g. Acidobacteria), the community as a whole was primarily driven by temporal changes over the growing season as distinct overwinter and crop-associated communities, with the same trend observed regardless of cropping system. Species-rich prokaryote communities were most stable over the growing season. Cropping system exerted a greater effect on fungal communities, with alpha-diversity highest and temporal changes most stable under CA. CON was particularly detrimental for alpha-diversity in AMF communities, with AMF alpha-diversity and stability improved under all other cropping systems. Practices that promoted alpha-diversity tended to also increase the similarity and temporal stability of soil fungal (and AMF) communities during a growing season, while prokaryote communities were largely insensitive to management., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Functional, physiological traits are the underlying drivers of niche differentiation. A common framework related to niches occupied by terrestrial prokaryotes is based on copiotrophy or oligotrophy, where resource investment is primarily in either rapid growth or stress tolerance, respectively. A quantitative trait-based approach sought relationships between taxa, traits and niche in terrestrial prokaryotes. With 175 taxa from 11 Phyla and 35 Families (n = 5 per Family), traits were considered as discrete counts of shared genome-encoded proteins. Trait composition strongly supported non-random functional distributions as preferential clustering of related taxa via unweighted pair-group method with arithmetic mean. Trait similarity between taxa increased as taxonomic rank decreased. A suite of Random Forest models identified traits significantly enriched or depleted in taxonomic groups. These traits conveyed functions related to rapid growth, nutrient acquisition and stress tolerance consistent with their presence in copiotroph-oligotroph niches. Hierarchical clustering of traits identified a clade of competitive, copiotrophic Families resilient to oxidative stress versus glycosyltransferase-enriched oligotrophic Families resistant to antimicrobials and environmental stress. However, the formation of five clades suggested a more nuanced view to describe niche differentiation in terrestrial systems is necessary. We suggest considering traits involved in both resource investment and acquisition when predicting niche., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS.)

Niche is a fundamental concept in ecology. It integrates the sum of biotic and abiotic environmental requirements that determines a taxon's distribution. Microbiologists currently lack quantitative approaches to address niche-related hypotheses. We tested four approaches for the quantification of niche breadth and overlap of taxa in amplicon sequencing datasets, with the goal of determining generalists, specialists and environmental-dependent distributions of community members. We applied these indices to in silico training datasets first, and then to real human gut and desert biological soil crust (biocrust) case studies, assessing the agreement of the indices with previous findings. Implementation of each approach successfully identified a priori conditions within in silico training data, and we found that by including a limit of quantification based on species rank, one could identify taxa falsely classified as specialists because of their low, sparse counts. Analysis of the human gut study offered quantitative support for Bacilli, Gammaproteobacteria and Fusobacteria specialists enriched after bariatric surgery. We could quantitatively characterise differential niche distributions of cyanobacterial taxa with respect to precipitation gradients in biocrusts. We conclude that these approaches, made publicly available as an R package (MicroNiche), represent useful tools to assess microbial environment-taxon and taxon-taxon relationships in a quantitative manner., (© FEMS 2020.)