Transcriptional chronology reveals conserved genes involved in pennate diatom sexual reproduction.

Sexual reproduction is a major driver of adaptation and speciation in eukaryotes. In diatoms, siliceous microalgae with a unique cell size reduction-restitution life cycle and among the world's most prolific primary producers, sex also acts as the main mechanism for cell size restoration through the formation of an expanding auxospore. However, the molecular regulators of the different stages of sexual reproduction and size restoration are poorly explored. Here, we combined RNA sequencing with the assembly of a 55 Mbp reference genome for Cylindrotheca closterium to identify patterns of gene expression during different stages of sexual reproduction. These were compared with a corresponding transcriptomic time series of Seminavis robusta to assess the degree of expression conservation. Integrative orthology analysis revealed 138 one-to-one orthologues that are upregulated during sex in both species, among which 56 genes consistently upregulated during cell pairing and gametogenesis, and 11 genes induced when auxospores are present. Several early, sex-specific transcription factors and B-type cyclins were also upregulated during sex in other pennate and centric diatoms, pointing towards a conserved core regulatory machinery for meiosis and gametogenesis across diatoms. Furthermore, we find molecular evidence that the pheromone-induced cell cycle arrest is short-lived in benthic diatoms, which may be linked to their active mode of mate finding through gliding. Finally, we exploit the temporal resolution of our comparative analysis to report the first marker genes for auxospore identity called AAE1-3 ("Auxospore-Associated Expression"). Altogether, we introduce a multi-species model of the transcriptional dynamics during size restoration in diatoms and highlight conserved gene expression dynamics during different stages of sexual reproduction.

Geology defines microbiome structure and composition in nunataks and valleys of the Sør Rondane Mountains, East Antarctica.

Understanding the relation between terrestrial microorganisms and edaphic factors in the Antarctic can provide insights into their potential response to environmental changes. Here we examined the composition of bacterial and micro-eukaryotic communities using amplicon sequencing of rRNA genes in 105 soil samples from the Sør Rondane Mountains (East Antarctica), differing in bedrock or substrate type and associated physicochemical conditions. Although the two most widespread taxa (Acidobacteriota and Chlorophyta) were relatively abundant in each sample, multivariate analysis and co-occurrence networks revealed pronounced differences in community structure depending on substrate type. In moraine substrates, Actinomycetota and Cercozoa were the most abundant bacterial and eukaryotic phyla, whereas on gneiss, granite and marble substrates, Cyanobacteriota and Metazoa were the dominant bacterial and eukaryotic taxa. However, at lower taxonomic level, a distinct differentiation was observed within the Cyanobacteriota phylum depending on substrate type, with granite being dominated by the Nostocaceae family and marble by the Chroococcidiopsaceae family. Surprisingly, metazoans were relatively abundant according to the 18S rRNA dataset, even in samples from the most arid sites, such as moraines in Austkampane and Widerøefjellet ("Dry Valley"). Overall, our study shows that different substrate types support distinct microbial communities, and that mineral soil diversity is a major determinant of terrestrial microbial diversity in inland Antarctic nunataks and valleys.

Visualisation of microalgal lipid bodies through electron microscopy.

In this study, transmission electron microscopy (TEM) and cryo-scanning electron microscopy (cryo-SEM) were evaluated for their ability to detect lipid bodies in microalgae. To do so, Phaeodactylum tricornutum and Nannochloropsis oculata cells were harvested in both the mid-exponential and early stationary growth phase. Two different cryo-SEM cutting methods were compared: cryo-planing and freeze-fracturing. The results showed that, despite the longer preparation time, TEM visualisation preceded by cryo-immobilisation allows a clear detection of lipid bodies and is preferable to cryo-SEM. Using freeze-fracturing, lipid bodies were rarely detected. This was only feasible if crystalline layers in the internal structure, most likely related to sterol esters or di-saturated triacylglycerols, were revealed. Furthermore, lipid bodies could not be detected using cryo-planing. Cryo-SEM is also not the preferred technique to recognise other organelles besides lipid bodies, yet it did reveal chloroplasts in both species and filament-containing organelles in cryo-planed Nannochloropsis oculata samples.

Polar lake microbiomes have distinct evolutionary histories.

Toward the poles, life on land is increasingly dominated by microorganisms, yet the evolutionary origin of polar microbiomes remains poorly understood. Here, we use metabarcoding of Arctic, sub-Antarctic, and Antarctic lacustrine benthic microbial communities to test the hypothesis that high-latitude microbiomes are recruited from a globally dispersing species pool through environmental selection. We demonstrate that taxonomic overlap between the regions is limited within most phyla, even at higher-order taxonomic levels, with unique deep-branching phylogenetic clades being present in each region. We show that local and regional taxon richness and net diversification rate of regionally restricted taxa differ substantially between polar regions in both microeukaryotic and bacterial biota. This suggests that long-term evolutionary divergence resulting from low interhemispheric dispersal and diversification in isolation has been a prominent process shaping present-day polar lake microbiomes. Our findings illuminate the distinctive biogeography of polar lake ecosystems and underscore that conservation efforts should include their unique microbiota.

Diatom adhesive trail proteins acquired by horizontal gene transfer from bacteria serve as primers for marine biofilm formation.

Biofilm-forming benthic diatoms are key primary producers in coastal habitats, where they frequently dominate sunlit intertidal substrata. The development of gliding motility in raphid diatoms was a key molecular adaptation that contributed to their evolutionary success. However, the structure-function correlation between diatom adhesives utilized for gliding and their relationship to the extracellular matrix that constitutes the diatom biofilm is unknown. Here, we have used proteomics, immunolocalization, comparative genomics, phylogenetics and structural homology analysis to investigate the evolutionary history and function of diatom adhesive proteins. Our study identified eight proteins from the adhesive trails of Craspedostauros australis, of which four form a new protein family called Trailins that contain an enigmatic Choice-of-Anchor A (CAA) domain, which was acquired through horizontal gene transfer from bacteria. Notably, the CAA-domain shares a striking structural similarity with one of the most widespread domains found in ice-binding proteins (IPR021884). Our work offers new insights into the molecular basis for diatom biofilm formation, shedding light on the function and evolution of diatom adhesive proteins. This discovery suggests that there is a transition in the composition of biomolecules required for initial surface colonization and those utilized for 3D biofilm matrix formation.

Structure Elucidation of the First Sex-Inducing Pheromone of a Diatom.

Diatoms are abundant unicellular microalgae, responsible for ≈20 % of global photosynthetic CO2 fixation. Nevertheless, we know little about fundamental aspects of their biology, such as their sexual reproduction. Pheromone-mediated chemical communication is crucial for successful mating. An attraction pheromone was identified in the diatom Seminavis robusta, but metabolites priming cells for sex and synchronizing search and mating behavior remained elusive. These sex-inducing pheromones (SIP) induce cell cycle arrest and trigger the production of the attraction pheromone. Here we describe the challenging structure elucidation of an S. robusta SIP. Guided by metabolomics, a candidate metabolite was identified and elucidated by labeling experiments, NMR, ESI MSn analyses, and chemical transformations. The use of negative ion mode MS was essential to decipher the unprecedented hydroxyproline and ß-sulfated aspartate-containing cyclic heptapeptide that acts in femtomolar concentrations.

Novel diversity of polar Cyanobacteria revealed by genome-resolved metagenomics.

Benthic microbial mats dominated by Cyanobacteria are important features of polar lakes. Although culture-independent studies have provided important insights into the diversity of polar Cyanobacteria, only a handful of genomes have been sequenced to date. Here, we applied a genome-resolved metagenomics approach to data obtained from Arctic, sub-Antarctic and Antarctic microbial mats. We recovered 37 metagenome-assembled genomes (MAGs) of Cyanobacteria representing 17 distinct species, most of which are only distantly related to genomes that have been sequenced so far. These include (i) lineages that are common in polar microbial mats such as the filamentous taxa Pseudanabaena, Leptolyngbya, Microcoleus/Tychonema and Phormidium; (ii) the less common taxa Crinalium and Chamaesiphon; (iii) an enigmatic Chroococcales lineage only distantly related to Microcystis; and (iv) an early branching lineage in the order Gloeobacterales that is distributed across the cold biosphere, for which we propose the name Candidatus Sivonenia alaskensis. Our results show that genome-resolved metagenomics is a powerful tool for expanding our understanding of the diversity of Cyanobacteria, especially in understudied remote and extreme environments.

Long-term phytoplankton dynamics in the Zeeschelde estuary (Belgium) are driven by the interactive effects of de-eutrophication, altered hydrodynamics and extreme weather events.

We studied how changing human impacts affected phytoplankton dynamics in the freshwater and brackish tidal reaches of the Zeeschelde estuary (Belgium) between 2002 and 2018. Until the early 2000s, the Zeeschelde was heavily polluted due to high wastewater discharges. By 2008, water quality had improved, resulting in lower nutrient concentrations and higher oxygen levels. Since 2009, however, increased dredging activities resulted in altered hydrodynamics and increased suspended sediment concentration. The combined effects of these environmental changes were reflected in three marked transitions in phytoplankton community composition. Assemblages were dominated by Thalassiosirales and green algae (especially Scenedesmaceae) until 2003. The period 2003-2011 was characterized by the wax and wane of the centric diatoms Actinocyclus and Aulacoseira, while in the period 2012-2018 Thalassiosirales and Cyanobacteria became dominant, the latter mainly imported from the tributaries. Phytoplankton biomass increased sharply in 2003, after which there was a gradual decline until 2018. By 2018, the timing of the growing season had advanced with about one month compared to the start of the study, probably as a consequence of climate warming and intensified zooplankton grazing pressure. Our study shows that de-eutrophication (during the 2000s) and morphological interventions in the estuary (in the 2010s) were dominant drivers of phytoplankton dynamics but that the main shifts in community composition were triggered by extreme weather events, suggesting significant resistance of autochthonous communities to gradual changes in the environment.

More than just hitchhikers: a survey of bacterial communities associated with diatoms originating from sea turtles.

Diatoms and bacteria are known for being the first colonizers of submerged surfaces including the skin of marine reptiles. Sea turtle carapace and skin harbor diverse prokaryotic and eukaryotic microbes, including several epizoic diatoms. However, the importance of diatom-bacteria associations is hardly investigated in biofilms associated with animal hosts. This study provides an inventory of diatoms, bacteria and diatom-associated bacteria originating from loggerhead sea turtles using both metabarcoding and culturing approaches. Amplicon sequencing of the carapace and skin samples chloroplast gene rbcL and 16S rRNA gene detected, in total, 634 diatom amplicon sequence variants (ASVs) and 3661 bacterial ASVs, indicating high diversity. Cultures of putative epizoic and non-epizoic diatoms contained 458 bacterial ASVs and their bacterial assemblages reflected those of their host. Diatom strains allowed for enrichment and isolation of bacterial families rarely observed on turtles, such as Marinobacteraceae, Alteromonadaceae and Alcanivoracaceae. When accounting for phylogenetic relationships between bacterial ASVs, we observed that related diatom genera might retain similar microbial taxa in culture, regardless of the turtle's skin or carapace source. These data provide deeper insights into the sea turtle-associated microbial communities, and reveal the potential of epizoic biofilms as a source of novel microbes and possibly important diatom-bacteria associations.

Selection constrains lottery assembly in the microbiomes of closely related diatom species.

It is generally recognised that interactions between microalgae and bacteria play an important role in the functioning of marine ecosystems. In this context, increasing attention is paid to the processes that shape microalga-associated microbiomes. In recent years, conflicting evidence has been reported with respect to the relative importance of selective vs neutral processes in the assembly process. Whereas some studies report strong selection imposed by the host, others propose a more neutral, lottery-like assembly model according to which the chance of bacteria becoming part of the microbiome is proportional to their abundance in the environment and not driven by the selectional pressure created by the host. In the present study, we investigated to what degree selective vs neutral assembly processes constrain taxonomic, phylogenetic and functional variation within and between microbiomes associated with 69 isolates belonging to the Cylindrotheca closterium benthic marine diatom complex. The diatom cultures were initiated from non-axenic clonal isolates from different marine environments and geographic locations, and were then reared in a common garden (lab) environment. An important environmental imprint, likely due to in situ lottery dynamics, was apparent in the diatom microbiomes. However, microbiome assembly was also phylogenetically and functionally constrained through selective filtering related to the host microhabitat. Randomised microbiome assembly simulations revealed evidence for phylogenetic overdispersion in the observed microbiomes, reflecting an important role in the assembly process for competition between bacteria on the one hand and predominantly genetically driven differences between the hosts on the other hand. Our study thus shows that even between closely related diatom strains, host selection affects microbiome assembly, superimposing the predominantly stochastically driven recruitment process.

Maximizing nutrient recycling from digestate for production of protein-rich microalgae for animal feed application.

The integration of phototrophic microalgal production and anaerobic digestion can recycle excess nutrients across European surplus hotspots to produce protein-rich biomass for nutritional applications. However, the challenging physico-chemical properties of raw digestate constrain microalgal growth and limit digestate valorization potential. This study focused on the pre-treatment of food waste-based digestate using paper-filtration to improve its properties for cultivating Desmodesmus sp. and Chlorella vulgaris. The microalgal growth performance in paper-filtered digestate (PFD, 10 µm-pore size) was then compared to growth in membrane-filtered digestate (MFD, 0.2 µm-pore size). A microplate-based screening coupled with Cytation device assessment of PFD and MFD samples after dilution and with/without phosphorus supplementation showed that PFD was the best substrate. Moreover, phosphorus supplementation resulted in improved growth at higher digestate concentrations (5-10% v/v PFD), indicating the importance of using a balanced growth medium to increase the volumetric usage of digestate. Results were validated in a 3-L bioreactor at 10% PFD with phosphorus supplementation, reaching a biomass concentration of 2.4 g L-1 with a protein and carbohydrate content of 67% and 13% w/w respectively. This trial indicates that paper-filtration is a promising pre-treatment technique to maximize digestate recycling and deliver a sustainable animal feed-grade protein alternative.

Light intensity and spectral composition drive reproductive success in the marine benthic diatom Seminavis robusta.

The properties of incident light play a crucial role in the mating process of diatoms, a group of ecologically important microalgae. While species-specific requirements for light intensity and photoperiod have been observed in several diatom species, little is known about the light spectrum that allows sexual reproduction. Here, we study the effects of spectral properties and light intensity on the initiation and progression of sexual reproduction in the model benthic diatom Seminavis robusta. We found that distinct stages of the mating process have different requirements for light. Vigorous mating pair formation occurred under a broad range of light intensities, ranging from 10 to 81 µE m-2 s-1, while gametogenesis and subsequent stages were strongly affected by moderate light intensities of 27 µE m-2 s-1 and up. In addition, light of blue or blue-green wavelengths was required for the formation of mating pairs. Combining flow cytometric analysis with expression profiling of the diatom-specific cyclin dsCyc2 suggests that progression through a blue light-dependent checkpoint in the G1 cell cycle phase is essential for induction of sexual reproduction. Taken together, we expand the current model of mating in benthic pennate diatoms, which relies on the interplay between light, cell cycle and sex pheromone signaling.

Extinction of austral diatoms in response to large-scale climate dynamics in Antarctica.

Despite evidence for microbial endemism, an understanding of the impact of geological and paleoclimate events on the evolution of regional protist communities remains elusive. Here, we provide insights into the biogeographical history of Antarctic freshwater diatoms, using lacustrine fossils from mid-Miocene and Quaternary Antarctica, and dovetail this dataset with a global inventory of modern freshwater diatom communities. We reveal the existence of a diverse mid-Miocene diatom flora bearing similarities with several former Gondwanan landmasses. Miocene cooling and Plio-Pleistocene glaciations triggered multiple extinction waves, resulting in the selective depauperation of this flora. Although extinction dominated, in situ speciation and new colonizations ultimately shaped the species-poor, yet highly adapted and largely endemic, modern Antarctic diatom flora. Our results provide a more holistic view on the scale of biodiversity turnover in Neogene and Pleistocene Antarctica than the fragmentary perspective offered by macrofossils and underscore the sensitivity of lacustrine microbiota to large-scale climate perturbations.

Mitotic recombination between homologous chromosomes drives genomic diversity in diatoms.

Diatoms, an evolutionarily successful group of microalgae, display high levels of intraspecific genetic variability in natural populations. However, the contribution of various mechanisms generating such diversity is unknown. Here we estimated the genetic micro-diversity within a natural diatom population and mapped the genomic changes arising within clonally propagated diatom cell cultures. Through quantification of haplotype diversity by next-generation sequencing and amplicon re-sequencing of selected loci, we documented a rapid accumulation of multiple haplotypes accompanied by the appearance of novel protein variants in cell cultures initiated from a single founder cell. Comparison of the genomic changes between mother and daughter cells revealed copy number variation and copy-neutral loss of heterozygosity leading to the fixation of alleles within individual daughter cells. The loss of heterozygosity can be accomplished by recombination between homologous chromosomes. To test this hypothesis, we established an endogenous readout system and estimated that the frequency of interhomolog mitotic recombination was under standard growth conditions 4.2 events per 100 cell divisions. This frequency is increased under environmental stress conditions, including treatment with hydrogen peroxide and cadmium. These data demonstrate that copy number variation and mitotic recombination between homologous chromosomes underlie clonal variability in diatom populations. We discuss the potential adaptive evolutionary benefits of the plastic response in the interhomolog mitotic recombination rate, and we propose that this may have contributed to the ecological success of diatoms.

Pheromone Mediated Sexual Reproduction of Pennate Diatom Cylindrotheca closterium.

Benthic diatoms dominate primary production in marine subtidal and intertidal environments. Their extraordinary species diversity and ecological success is thought to be linked with their predominantly heterothallic sexual reproduction. Little is known about pheromone involvement during mating of pennate diatoms. Here we describe pheromone guided mating in the coastal raphid diatom Cylindrotheca closterium. We show that the two mating types (mt+ and mt-) have distinct functions. Similar to other benthic diatoms, mt+ cells are searching for the mt- cells to pair. To enhance mating efficiency mt- exudes an attraction pheromone which we proved by establishing a novel capillary assay. Further, two more pheromones produced by mt- promote the sexual events. One arrests the cell cycle progression of mt+ while the other induces gametogenesis of mt+. We suggest that C. closterium shares a functionally similar pheromone system with other pennate diatoms like Seminavis robusta and Pseudostaurosira trainorii which synchronize sexual events and mate attraction. Remarkably, we found no evidence of mt+ producing pheromones, which differentiates C. closterium from other pennates and suggests a less complex pheromone system in C. closterium.

Diurnal transcript profiling of the diatom Seminavis robusta reveals adaptations to a benthic lifestyle.

Coastal regions contribute an estimated 20% of annual gross primary production in the oceans, despite occupying only 0.03% of their surface area. Diatoms frequently dominate coastal sediments, where they experience large variations in light regime resulting from the interplay of diurnal and tidal cycles. Here, we report on an extensive diurnal transcript profiling experiment of the motile benthic diatom Seminavis robusta. Nearly 90% (23 328) of expressed protein-coding genes and 66.9% (1124) of expressed long intergenic non-coding RNAs showed significant expression oscillations and are predominantly phasing at night with a periodicity of 24 h. Phylostratigraphic analysis found that rhythmic genes are enriched in highly conserved genes, while diatom-specific genes are predominantly associated with midnight expression. Integration of genetic and physiological cell cycle markers with silica depletion data revealed potential new silica cell wall-associated gene families specific to diatoms. Additionally, we observed 1752 genes with a remarkable semidiurnal (12-h) periodicity, while the expansion of putative circadian transcription factors may reflect adaptations to cope with highly unpredictable external conditions. Taken together, our results provide new insights into the adaptations of diatoms to the benthic environment and serve as a valuable resource for the study of diurnal regulation in photosynthetic eukaryotes.

Altering the Sex Pheromone Cyclo(l-Pro-l-Pro) of the Diatom Seminavis robusta towards a Chemical Probe.

As a major group of algae, diatoms are responsible for a substantial part of the primary production on the planet. Pennate diatoms have a predominantly benthic lifestyle and are the most species-rich diatom group, with members of the raphid clades being motile and generally having heterothallic sexual reproduction. It was recently shown that the model species Seminavis robusta uses multiple sexual cues during mating, including cyclo(l-Pro-l-Pro) as an attraction pheromone. Elaboration of the pheromone-detection system is a key aspect in elucidating pennate diatom life-cycle regulation that could yield novel fundamental insights into diatom speciation. This study reports the synthesis and bio-evaluation of seven novel pheromone analogs containing small structural alterations to the cyclo(l-Pro-l-Pro) pheromone. Toxicity, attraction, and interference assays were applied to assess their potential activity as a pheromone. Most of our analogs show a moderate-to-good bioactivity and low-to-no phytotoxicity. The pheromone activity of azide- and diazirine-containing analogs was unaffected and induced a similar mating behavior as the natural pheromone. These results demonstrate that the introduction of confined structural modifications can be used to develop a chemical probe based on the diazirine- and/or azide-containing analogs to study the pheromone-detection system of S. robusta.

Mating type specific transcriptomic response to sex inducing pheromone in the pennate diatom Seminavis robusta.

Sexual reproduction is a fundamental phase in the life cycle of most diatoms. Despite its role as a source of genetic variation, it is rarely reported in natural circumstances and its molecular foundations remain largely unknown. Here, we integrate independent transcriptomic datasets to prioritize genes responding to sex inducing pheromones (SIPs) in the pennate diatom Seminavis robusta. We observe marked gene expression changes associated with SIP treatment in both mating types, including an inhibition of S phase progression, chloroplast division, mitosis, and cell wall formation. Meanwhile, meiotic genes are upregulated in response to SIP, including a sexually induced diatom specific cyclin. Our data further suggest an important role for reactive oxygen species, energy metabolism, and cGMP signaling during the early stages of sexual reproduction. In addition, we identify several genes with a mating type specific response to SIP, and link their expression pattern with physiological specialization, such as the production of the attraction pheromone diproline in mating type - (MT-) and mate-searching behavior in mating type + (MT+). Combined, our results provide a model for early sexual reproduction in pennate diatoms and significantly expand the suite of target genes to detect sexual reproduction events in natural diatom populations.

Author Correction: The Seminavis robusta genome provides insights into the evolutionary adaptations of benthic diatoms.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The Seminavis robusta genome provides insights into the evolutionary adaptations of benthic diatoms.

Benthic diatoms are the main primary producers in shallow freshwater and coastal environments, fulfilling important ecological functions such as nutrient cycling and sediment stabilization. However, little is known about their evolutionary adaptations to these highly structured but heterogeneous environments. Here, we report a reference genome for the marine biofilm-forming diatom Seminavis robusta, showing that gene family expansions are responsible for a quarter of all 36,254 protein-coding genes. Tandem duplications play a key role in extending the repertoire of specific gene functions, including light and oxygen sensing, which are probably central for its adaptation to benthic habitats. Genes differentially expressed during interactions with bacteria are strongly conserved in other benthic diatoms while many species-specific genes are strongly upregulated during sexual reproduction. Combined with re-sequencing data from 48 strains, our results offer insights into the genetic diversity and gene functions in benthic diatoms.