Paleo-diatom composition from Santa Barbara Basin deep-sea sediments: a comparison of 18S-V9 and diat-rbcL metabarcoding vs shotgun metagenomics

Sedimentary ancient DNA (sedaDNA) analyses are increasingly used to reconstruct marine ecosystems. The majority of marine sedaDNA studies use a metabarcoding approach (extraction and analysis of specific DNA fragments of a defined length), targeting short taxonomic marker genes. Promising examples are 18S-V9 rRNA (~121–130 base pairs, bp) and diat-rbcL (76 bp), targeting eukaryotes and diatoms, respectively. However, it remains unknown how 18S-V9 and diat-rbcL derived compositional profiles compare to metagenomic shotgun data, the preferred method for ancient DNA analyses as amplification biases are minimised. We extracted DNA from five Santa Barbara Basin sediment samples (up to ~11 000 years old) and applied both a metabarcoding (18S-V9 rRNA, diat-rbcL) and a metagenomic shotgun approach to (i) compare eukaryote, especially diatom, composition, and (ii) assess sequence length and database related biases. Eukaryote composition differed considerably between shotgun and metabarcoding data, which was related to differences in read lengths (~112 and ~161 bp, respectively), and overamplification of short reads in metabarcoding data. Diatom composition was influenced by reference bias that was exacerbated in metabarcoding data and characterised by increased representation of Chaetoceros, Thalassiosira and Pseudo-nitzschia. Our results are relevant to sedaDNA studies aiming to accurately characterise paleo-ecosystems from either metabarcoding or metagenomic data.

Gynostemium morphology and floral biology of Thottea duchartrei Sivar., A.Babu & Balach. (Aristolochiaceae), an endemic species from Western Ghats, India

Gynostemium is a putative floral structure formed by the fusion of the male and female reproductive parts (stamens and carpels) through a process during its development called synorganization. Most of the Indian species of Thottea Rottb. have a prominent gynostemium in their floral architecture, with varying degrees of adnation and complexity. Even though the gynostemium is a significant taxonomic marker for the inter-specific systematics in Thottea, the scepticism and arguments on deciphering its morphology and functional parameters, including the existence of staminodes, makes it imperative to be examined in detail. The present study is the first attempt in this line which aims to explore the morphological characters of gynostemium and reproductive biology of Thottea duchartrei Sivar., A.Babu and Balach., an endemic species in the Western Ghats. Stereo zoom microscopy, SEM and reproductive biological studies were conducted providing special emphasis to the gynostemium. Detailed analysis revealed the structural and functional diversity of gynostemium with regard to its components. The study also helped to recognize the columnar outgrowths on the gynostemium as staminodes. In total, the present study helps to solve the confusions regarding the functional identity of the gynostemium T. duchartrei with regard to its morphology.

Trait-trait relationships and functional tradeoffs vary with genome size in prokaryotes

We report genomic traits that have been associated with the life history of prokaryotes and highlight conflicting findings concerning earlier observed trait correlations and tradeoffs. In order to address possible explanations for these contradictions we examined trait-trait variations of 11 genomic traits from ~ 17,000 sequenced genomes. The studied trait-trait variations suggested: (i) the predominance of two resistance and resilience-related orthogonal axes , (ii) an overlap between a resilience axis and an axis of resource usage efficiency. These findings imply that resistance associated traits of prokaryotes are globally decoupled from resilience and resource use efficiencies associated traits. However, further inspection of pairwise scatterplots showed that resistance and resilience traits tended to be positively related for genomes up to roughly five million base pairs and negatively for larger genomes. This in turn precludes a globally consistent assignment of prokaryote genomic traits to the competitor - stress-tolerator -ruderal (CSR) schema that sorts species depending on their location along disturbance and productivity gradients into three ecological strategies and may serve as an explanation for conflicting findings from earlier studies. All reviewed genomic traits featured significant phylogenetic signals and we propose that our trait table can be applied to extrapolate genomic traits from taxonomic marker genes. This will enable to empirically evaluate the assembly of these genomic traits in prokaryotic communities from different habitats and under different productivity and disturbance scenarios as predicted via the resistance-resilience framework formulated here.

Extended taxonomic profiling of diverse environments with mOTUs 2.6

SummaryIdentifying species and estimating their relative abundance in metagenomic samples is a crucial task in microbiome research. However, for many environments, variable fractions of microbial species are not represented in reference genome databases, and thus, remain often unaccounted for in taxonomic composition analyses. Here, we present an updated version of the metagenomic profiling tool mOTUs. We extended its database of taxonomic marker genes by including information from ~600,000 prokaryotic draft genomes, >96% of which are metagenome assembled genomes from 23 environments. This extension enables researchers to profile a previously underrepresented diversity of microbes not only in some of the most intensively studied environments such as the human, soil and ocean microbiomes, but also in freshwater systems, the air, and the gastrointestinal tract of mice, cattle and other animals. This update doubles the number of detectable prokaryotic species to 33,570 and includes the release of 11,164 taxonomic profiles as a community resource.Availability and implementationmOTUs 2.6 is implemented in Python and licensed under GPLv3. Source code, profiles and documentation are available at: https://motu-tool.org/.Supplementary informationSupplementary information is available online.

Determinants of Staphylococcus aureus carriage in the developing infant nasal microbiome

Background Staphylococcus aureus is a leading cause of healthcare- and community-associated infections and can be difficult to treat due to antimicrobial resistance. About 30% of individuals carry S. aureus asymptomatically in their nares, a risk factor for later infection, and interactions with other species in the nasal microbiome likely modulate its carriage. It is thus important to identify ecological or functional genetic elements within the maternal or infant nasal microbiomes that influence S. aureus acquisition and retention in early life. Results We recruited 36 mother-infant pairs and profiled a subset of monthly longitudinal nasal samples from the first year after birth using shotgun metagenomic sequencing. The infant nasal microbiome is highly variable, particularly within the first 2 months. It is weakly influenced by maternal nasal microbiome composition, but primarily shaped by developmental and external factors, such as daycare. Infants display distinctive patterns of S. aureus carriage, positively associated with Acinetobacter species, Streptococcus parasanguinis, Streptococcus salivarius, and Veillonella species and inversely associated with maternal Dolosigranulum pigrum. Furthermore, we identify a gene family, likely acting as a taxonomic marker for an unclassified species, that is significantly anti-correlated with S. aureus in infants and mothers. In gene content-based strain profiling, infant S. aureus strains are more similar to maternal strains. Conclusions This improved understanding of S. aureus colonization is an important first step toward the development of novel, ecological therapies for controlling S. aureus carriage.

Gene exchange networks define species-like units in marine prokaryotes

SUMMARYAlthough horizontal gene transfer is recognized as a major evolutionary process in Bacteria and Archaea, its general patterns remain elusive, due to difficulties tracking genes at relevant resolution and scale within complex microbiomes. To circumvent these challenges, we analyzed a randomized sample of >12,000 genomes of individual cells of Bacteria and Archaea in the tropical and subtropical ocean - a well-mixed, global environment. We found that marine microorganisms form gene exchange networks (GENs) within which transfers of both flexible and core genes are frequent, including the rRNA operon that is commonly used as a conservative taxonomic marker. The data revealed efficient gene exchange among genomes with <28% nucleotide difference, indicating that GENs are much broader lineages than the nominal microbial species, which are currently delineated at 4-6% nucleotide difference. The 42 largest GENs accounted for 90% of cells in the tropical ocean microbiome. Frequent gene exchange within GENs helps explain how marine microorganisms maintain millions of rare genes and adapt to a dynamic environment despite extreme genome streamlining of their individual cells. Our study suggests that sharing of pangenomes through horizontal gene transfer is a defining feature of fundamental evolutionary units in marine planktonic microorganisms and, potentially, other microbiomes.

ARDEP, a Rapid Degenerate Primer Design Pipeline Based on k-mers for Amplicon Microbiome Studies

The survey of microbial diversity in various environments has relied upon the widespread use of well-evaluated amplification primers for taxonomic marker genes (e.g., prokaryotic 16S and fungal ITS). However, it is urgent to develop a fast and accurate bioinformatic program to design primers for microbial functional genes to explore more mechanisms in the microbial community. Here, we provide a rapid degenerate primer design pipeline (ARDEP) based on the k-mer algorithm, which can bypass the time-consuming step of sequence alignment to greatly reduce run times while ensuring accuracy. In addition, we developed an open-access platform for the implementation of primer design projects that could also calculate the amplification product length, GC content, Annealing Temperature (Tm), and ΔG of primer self-folding, and identify covered species and functional groups. Using this new platform, we designed primers for several functional genes in the nitrogen cycle, including napA and amoA. Our newly designed primers achieved higher coverage than the commonly used primers for all tested genes. The program and the associated platform that applied the k-mer algorithm could greatly enhance the design and evaluation of primers for environmental microbiome studies.

The Impact of Primer Design on Amplicon-Based Metagenomic Profiling Accuracy: Detailed Insights into Bifidobacterial Community Structure

Next Generation Sequencing (NGS) technologies have overcome the limitations of cultivation-dependent approaches and allowed detailed study of bacterial populations that inhabit the human body. The consortium of bacteria residing in the human intestinal tract, also known as the gut microbiota, impacts several physiological processes important for preservation of the health status of the host. The most widespread microbiota profiling method is based on amplification and sequencing of a variable portion of the 16S rRNA gene as a universal taxonomic marker among members of the Bacteria domain. Despite its popularity and obvious advantages, this 16S rRNA gene-based approach comes with some important limitations. In particular, the choice of the primer pair for amplification plays a major role in defining the accuracy of the reconstructed bacterial profiles. In the current study, we performed an in silico PCR using all currently described 16S rRNA gene-targeting primer pairs (PP) in order to assess their efficiency. Our results show that V3, V4, V5, and V6 were the optimal regions on which to design 16S rRNA metagenomic primers. In detail, PP39 (Probio_Uni/Probio_Rev), PP41 (341F/534R), and PP72 (970F/1050R) were the most suitable primer pairs with an amplification efficiency of >98.5%. Furthermore, the Bifidobacterium genus was examined as a test case for accurate evaluation of intra-genus performances at subspecies level. Intriguingly, the in silico analysis revealed that primer pair PP55 (527f/1406r) was unable to amplify the targeted region of any member of this bacterial genus, while several other primer pairs seem to rather inefficiently amplify the target region of the main bifidobacterial taxa. These results highlight that selection of a 16S rRNA gene-based PP should be done with utmost care in order to avoid biases in microbiota profiling results.

A Genus Definition for Bacteria and Archaea Based on a Standard Genome Relatedness Index

ABSTRACT Genus assignment is fundamental in the characterization of microbes, yet there is currently no unambiguous way to demarcate genera solely using standard genomic relatedness indices. Here, we propose an approach to demarcate genera that relies on the combined use of the average nucleotide identity, genome alignment fraction, and the distinction between type- and non-type species. More than 3,500 genomes representing type strains of species from >850 genera of either bacterial or archaeal lineages were tested. Over 140 genera were analyzed in detail within the taxonomic context of order/family. Significant genomic differences between members of a genus and type species of other genera in the same order/family were conserved in 94% of the cases. Nearly 90% (92% if polyphyletic genera are excluded) of the type strains were classified in agreement with current taxonomy. The 448 type strains that need reclassification directly impact 33% of the genera analyzed in detail. The results provide a first line of evidence that the combination of genomic indices provides added resolution to effectively demarcate genera within the taxonomic framework that is currently based on the 16S rRNA gene. We also identify the emergence of natural breakpoints at the genome level that can further help in the circumscription of taxa, increasing the proportion of directly impacted genera to at least 43% and pointing at inaccuracies on the use of the 16S rRNA gene as a taxonomic marker, despite its precision. Altogether, these results suggest that genomic coherence is an emergent property of genera in Bacteria and Archaea. IMPORTANCE In recent decades, the taxonomy of Bacteria and Archaea, and therefore genus designation, has been largely based on the use of a single ribosomal gene, the 16S rRNA gene, as a taxonomic marker. We propose an approach to delineate genera that excludes the direct use of the 16S rRNA gene and focuses on a standard genome relatedness index, the average nucleotide identity. Our findings are of importance to the microbiology community because the emergent properties of Bacteria and Archaea that are identified in this study will help assign genera with higher taxonomic resolution.

Pollen morphology and its systematic implication on some species of Artemisia L. from Gilgit-Baltistan Pakistan

This study was accomplished to scrutinize the pollen morphology of 15 species of the genus Artemisia of the family Asteraceae from Gilgit-Baltistan region of Pakistan by means of scanning electron microscopy (SEM). Results revealed pollen grains of Artemisia species with tricolporate shape, and characterized by globular symmetry (ellipsoid ball shaped from equatorial side and three lobed rounds from polar view) with few exceptions. Additionally, the pollens are marked with reduced spinules on their surfaces which are diagnostic character for the genus Artemisia. In this study, seven micromorphological characters of pollen grains of 15 Artemisia species viz. shape of pollen, arrangement of spinules, exine sculpture, spinules base, equatorial width and polar length, were employed to construct a dendrogram following the consequential cluster analyses. In the dentrogram, four groups within the studied Artemisia species have been recognized. The pollen morphology of Artemisia could be a good taxonomic marker to cope with its taxonomic delimitations in combination with other floral and molecular attributes.