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.

Gene Duplication, Shifting Selection, and Dosage Balance of Silicon Transporter Proteins in Marine and Freshwater Diatoms.

Numerous factors shape the evolution of protein-coding genes, including shifts in the strength or type of selection following gene duplications or changes in the environment. Diatoms and other silicifying organisms use a family of silicon transporters (SITs) to import dissolved silicon from the environment. Freshwaters contain higher silicon levels than oceans, and marine diatoms have more efficient uptake kinetics and less silicon in their cell walls, making them better competitors for a scarce resource. We compiled SITs from 37 diatom genomes to characterize shifts in selection following gene duplications and marine-freshwater transitions. A deep gene duplication, which coincided with a whole-genome duplication, gave rise to two gene lineages. One of them (SIT1-2) is present in multiple copies in most species and is known to actively import silicon. These SITs have evolved under strong purifying selection that was relaxed in freshwater taxa. Episodic diversifying selection was detected but not associated with gene duplications or habitat shifts. In contrast, genes in the second SIT lineage (SIT3) were present in just half the species, the result of multiple losses. Despite conservation of SIT3 in some lineages for the past 90-100 million years, repeated losses, relaxed selection, and low expression highlighted the dispensability of SIT3, consistent with a model of deterioration and eventual loss due to relaxed selection on SIT3 expression. The extensive but relatively balanced history of duplications and losses, together with paralog-specific expression patterns, suggest diatoms continuously balance gene dosage and expression dynamics to optimize silicon transport across major environmental gradients.

Resolving Marine-Freshwater Transitions by Diatoms Through a Fog of Gene Tree Discordance.

Despite the obstacles facing marine colonists, most lineages of aquatic organisms have colonized and diversified in freshwaters repeatedly. These transitions can trigger rapid morphological or physiological change and, on longer timescales, lead to increased rates of speciation and extinction. Diatoms are a lineage of ancestrally marine microalgae that have diversified throughout freshwater habitats worldwide. We generated a phylogenomic data set of genomes and transcriptomes for 59 diatom taxa to resolve freshwater transitions in one lineage, the Thalassiosirales. Although most parts of the species tree were consistently resolved with strong support, we had difficulties resolving a Paleocene radiation, which affected the placement of one freshwater lineage. This and other parts of the tree were characterized by high levels of gene tree discordance caused by incomplete lineage sorting and low phylogenetic signal. Despite differences in species trees inferred from concatenation versus summary methods and codons versus amino acids, traditional methods of ancestral state reconstruction supported six transitions into freshwaters, two of which led to subsequent species diversification. Evidence from gene trees, protein alignments, and diatom life history together suggest that habitat transitions were largely the product of homoplasy rather than hemiplasy, a condition where transitions occur on branches in gene trees not shared with the species tree. Nevertheless, we identified a set of putatively hemiplasious genes, many of which have been associated with shifts to low salinity, indicating that hemiplasy played a small but potentially important role in freshwater adaptation. Accounting for differences in evolutionary outcomes, in which some taxa became locked into freshwaters while others were able to return to the ocean or become salinity generalists, might help further distinguish different sources of adaptive mutation in freshwater diatoms.

Diatom endemism and taxonomic turnover: Assessment in high-altitude alpine lakes covering a large geographical range.

Diatoms are widely used as ecological indicators and show various degrees of endemism. Many studies that support the idea of endemic species integrate several climate zones, a variety of ecosystem types, and often focus on a global scale. Here, we investigated whether endemism could be detected when considering a homogeneous type of ecosystem in a single climate zone. We sampled stone biofilms at 40-50 cm depth in high-altitude lakes in the Alpine climate zone. A total of 149 samples were obtained from the French and Georgian mountains, two areas separated by ∼3000 km. Using Amplicon Sequence Variants derived from DNA metabarcoding, we assessed taxonomic turnover and Zeta-diversity (a measure of endemism). We ran haplotype networks and phylogenetic tests to measure geographical signal in the phylogenies of dominant taxa. The French and Georgian communities shared 51 % of species. Species that were not shared across both regions were mostly rare, and often not characteristic of lakes but of neighboring habitats instead. In contrast, at the sub-species level, 87 % of the genotypes showed restricted distributions. Whereas endemism was the rule at sub-species level, most species were shared across both French and Georgian lakes, suggesting that geographic barriers strongly limited dispersal at the sub-species level but not species level. Dominant species hosted higher levels of sub-specific diversity than rare species. In contrast to global-scale studies, we did not find any significant geographical structuring in the phylogeny of the investigated species. This could indicate ongoing dispersal at a frequency fast enough to prevent allopatric divergence, yet slow enough to prevent sharing most haplotypes between France and Georgia. These results have implications for biomonitoring: depending on the taxonomic level chosen, robust generic tools (species level) or tools dedicated to a region able to discriminate fine pressures differences (sub-species level) may be developed.

The dynamic response to hypo-osmotic stress reveals distinct stages of freshwater acclimation by a euryhaline diatom.

The salinity gradient separating marine and freshwater environments is a major ecological divide, and the mechanisms by which most organisms adapt to new salinity environments are poorly understood. Diatoms are a lineage of ancestrally marine microalgae that have repeatedly colonized and diversified in freshwaters. Cyclotella cryptica is a euryhaline diatom found in salinities ranging from fully freshwater to fully marine, thus providing a powerful system for understanding the genomic mechanisms for mitigating and acclimating to low salinity. To understand how diatoms mitigate acute hypo-osmotic stress, we abruptly shifted C. cryptica from seawater to freshwater and performed transcriptional profiling during the first 10 h. Freshwater shock dramatically remodelled the transcriptome, with ~50% of the genome differentially expressed in at least one time point. The peak response occurred within 1 h, with strong repression of genes involved in cell growth and osmolyte production, and strong induction of specific stress defence genes. Transcripts largely returned to baseline levels within 4-10 h, with growth resuming shortly thereafter, suggesting that gene expression dynamics may be useful for predicting acclimation. Moreover, comparison to a transcriptomics study of C. cryptica following months-long acclimation to freshwater revealed little overlap between the genes and processes differentially expressed in cells exposed to acute stress versus fully acclimated conditions. Altogether, this study highlights the power of time-resolved transcriptomics to reveal fundamental insights into how cells dynamically respond to an acute environmental shift and provides new insights into how diatoms mitigate natural salinity fluctuations and have successfully diversified across freshwater habitats worldwide.

Strain-specific transcriptional responses overshadow salinity effects in a marine diatom sampled along the Baltic Sea salinity cline.

The salinity gradient separating marine and freshwater environments represents a major ecological divide for microbiota, yet the mechanisms by which marine microbes have adapted to and ultimately diversified in freshwater environments are poorly understood. Here, we take advantage of a natural evolutionary experiment: the colonization of the brackish Baltic Sea by the ancestrally marine diatom Skeletonema marinoi. To understand how diatoms respond to low salinity, we characterized transcriptomic responses of acclimated S. marinoi grown in a common garden. Our experiment included eight strains from source populations spanning the Baltic Sea salinity cline. Gene expression analysis revealed that low salinities induced changes in the cellular metabolism of S. marinoi, including upregulation of photosynthesis and storage compound biosynthesis, increased nutrient demand, and a complex response to oxidative stress. However, the strain effect overshadowed the salinity effect, as strains differed significantly in their response, both regarding the strength and the strategy (direction of gene expression) of their response. The high degree of intraspecific variation in gene expression observed here highlights an important but often overlooked source of biological variation associated with how diatoms respond to environmental change.

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.

Global radiation in a rare biosphere soil diatom.

Soil micro-organisms drive the global carbon and nutrient cycles that underlie essential ecosystem functions. Yet, we are only beginning to grasp the drivers of terrestrial microbial diversity and biogeography, which presents a substantial barrier to understanding community dynamics and ecosystem functioning. This is especially true for soil protists, which despite their functional significance have received comparatively less interest than their bacterial counterparts. Here, we investigate the diversification of Pinnularia borealis, a rare biosphere soil diatom species complex, using a global sampling of >800 strains. We document unprecedented high levels of species-diversity, reflecting a global radiation since the Eocene/Oligocene global cooling. Our analyses suggest diversification was largely driven by colonization of novel geographic areas and subsequent evolution in isolation. These results illuminate our understanding of how protist diversity, biogeographical patterns, and members of the rare biosphere are generated, and suggest allopatric speciation to be a powerful mechanism for diversification of micro-organisms.

Urbanization drives cross-taxon declines in abundance and diversity at multiple spatial scales.

The increasing urbanization process is hypothesized to drastically alter (semi-)natural environments with a concomitant major decline in species abundance and diversity. Yet, studies on this effect of urbanization, and the spatial scale at which it acts, are at present inconclusive due to the large heterogeneity in taxonomic groups and spatial scales at which this relationship has been investigated among studies. Comprehensive studies analysing this relationship across multiple animal groups and at multiple spatial scales are rare, hampering the assessment of how biodiversity generally responds to urbanization. We studied aquatic (cladocerans), limno-terrestrial (bdelloid rotifers) and terrestrial (butterflies, ground beetles, ground- and web spiders, macro-moths, orthopterans and snails) invertebrate groups using a hierarchical spatial design, wherein three local-scale (200 m × 200 m) urbanization levels were repeatedly sampled across three landscape-scale (3 km × 3 km) urbanization levels. We tested for local and landscape urbanization effects on abundance and species richness of each group, whereby total richness was partitioned into the average richness of local communities and the richness due to variation among local communities. Abundances of the terrestrial active dispersers declined in response to local urbanization, with reductions up to 85% for butterflies, while passive dispersers did not show any clear trend. Species richness also declined with increasing levels of urbanization, but responses were highly heterogeneous among the different groups with respect to the richness component and the spatial scale at which urbanization impacts richness. Depending on the group, species richness declined due to biotic homogenization and/or local species loss. This resulted in an overall decrease in total richness across groups in urban areas. These results provide strong support to the general negative impact of urbanization on abundance and species richness within habitat patches and highlight the importance of considering multiple spatial scales and taxa to assess the impacts of urbanization on biodiversity.

Extensive Cryptic Diversity in the Terrestrial Diatom Pinnularia borealis (Bacillariophyceae).

With the increasing application of molecular techniques for diatom species discovery and identification, it is important both from a taxonomic as well as an ecological and applied perspective, to understand in which groups morphological species delimitation is congruent with molecular approaches, or needs reconsideration. Moreover, such studies can improve our understanding of morphological trait evolution in this important group of microalgae. In this study, we used morphometric analysis on light microscopy (LM) micrographs in SHERPA, detailed scanning electron microscopy (SEM), and cytological observations in LM to examine 70 clones belonging to eight distinct molecular lineages of the cosmopolitan terrestrial diatom Pinnularia borealis. Due to high within-lineage variation, no conclusive morphological separation in LM nor SEM could be detected. Morphological stasis due to the "low-morphology" problem or stabilizing selection, as well as parallel/convergent evolution, phenotypic plasticity and structural inheritance are discussed as potential drivers for the observations. Altogether, P. borealis is truly cryptic, in contrast to the majority of other diatom species complexes which turned out to be pseudo-cryptic following detailed morphological analysis.

A polyphasic approach to the delimitation of diatom species: a case study for the genus Pinnularia (Bacillariophyta).

Diatoms are one of the most abundant and arguably the most species-rich group of protists. Diatom species delimitation has often been based exclusively on the recognition of morphological discontinuities without investigation of other lines of evidence. Even though DNA sequences and reproductive experiments have revealed several examples of (pseudo)cryptic diversity, our understanding of diatom species boundaries and diversity remains limited. The cosmopolitan pennate raphid diatom genus Pinnularia represents one of the most taxon-rich diatom genera. In this study, we focused on the delimitation of species in one of the major clades of the genus, the Pinnularia subgibba group, based on 105 strains from a worldwide origin. We compared genetic distances between the sequences of seven molecular markers and selected the most variable pair, the mitochondrial cox1 and nuclear encoded LSU rDNA, to formulate a primary hypothesis on the species limits using three single-locus automated species delimitation methods. We compared the DNA-based primary hypotheses with morphology and with other available lines of evidence. The results indicate that our data set comprised 15 species of the P. subgibba group. The vast majority of these taxa have an uncertain taxonomic identity, suggesting that several may be unknown to science and/or members of (pseudo)cryptic species complexes within the P. subgibba group.

Incomplete Reproductive Isolation Between Genetically Distinct Sympatric Clades of the Pennate Model Diatom Seminavis robusta.

Incomplete reproductive isolation between genetically distinct taxa provides an interesting opportunity for speciation and adaptation studies. This phenomenon is well-described in macro-organisms, but less experimental evidence is available for unicellular eukaryotes. Here, we document the sympatric occurrence of genetically differentiated populations of the pennate model diatom Seminavis robusta in coastal subtidal biofilm communities and show widespread potential for gene flow between them. Based on sequence variation in the plastid-encoded rbcL gene, three distinct clades were identified. Morphological variation between the clades reflected their phylogenetic relationships, with subtle differences in valve morphology in the most distant clade compared to the other two clades, which were indistinguishable. Using a large number of experimental crosses we showed that, although reproductive output was significantly lower compared to the majority of within-clade crosses, approximately 34.5% of the inter-clade crosses resulted in viable and fertile progeny. While the nature of the incomplete reproductive isolation remains unknown, its occurrence in natural diatom populations represents an additional mechanism contributing to population genetic structuring and adaptation and can spur further research into the mechanisms of species divergence and the maintenance of species identity in the presence of gene flow.

Expanding the toolbox for cryopreservation of marine and freshwater diatoms.

Diatoms constitute the most diverse group of microalgae and have long been recognised for their large biotechnological potential. In the wake of growing research interest in new model species and development of commercial applications, there is a pressing need for long-term preservation of diatom strains. While cryopreservation using dimethylsulfoxide (DMSO) as a cryoprotective agent is the preferred method for long-term strain preservation, many diatom species cannot be successfully cryopreserved using DMSO. Therefore, in this study, we studied cryopreservation success in six different diatom species, representing the major morphological and ecological diatom groups, using a range of DMSO concentrations and Plant Vitrification Solution 2 (PVS2) as an alternative cryoprotectant to DMSO. In addition, we tested whether suppressing bacterial growth by antibiotics accelerates the post-thaw recovery process. Our results show that the effects of cryoprotectant choice, its concentration and the addition of antibiotics are highly species specific. In addition, we showed that PVS2 and antibiotics are useful agents to optimize cryopreservation of algae that cannot survive the traditional cryopreservation protocol using DMSO. We conclude that a species-specific approach will remain necessary to develop protocols for diatom cryopreservation and to increase their representation in public culture collections.