Metabarcoding of environmental DNA is widely used to assess fungal diversity, yet the choice of bioinformatic pipeline strongly influences results. Here we compare five pipelines to detect Cladonia diversity from soil eDNA across Europe, using an extensive floristic survey combined with Sanger sequencing as a reference. All pipelines similarly detected dominant species but differed in their recovery of rare taxa, hidden diversity, or putative artefacts. The total number of operational taxonomic units (OTUs) varied 2.5-fold across pipelines, and overlap with the reference dataset ranged from 21% to 40%. The pipeline by Bálint et al. (2014), followed by Swarm v2 clustering and VSEARCH chimera filtering, achieved the greatest agreement with the taxonomic survey and recovered the highest number of rare OTUs. The DADA2-based pipeline with DECIPHER clustering yielded the highest number of OTUs not found by the taxonomic survey. In contrast, LULU curation yielded the most conservative dataset, with the lowest diversity and lowest overlap with the Sanger-verified reference.

Abstract The processes underlying microbial diversity are inherently complex, so it is highly challenging to establish universal explanations and theoretical models of speciation. Using a population genetic approach, our study reveals that even closely related protist taxa with similar distributions, inhabiting overlapping ecological niches and comparable abundances, employ distinct speciation strategies. We investigated the population structure of two related species, Synura petersenii and Synura glabra (Chrysophyceae, Stramenopiles), across Europe. Using single-digest restriction-site-associated sequencing, we analyzed genomes of both species from a total of 43 distinct water bodies. A population pattern was present in both algal species, demonstrating their recent divergence and ongoing speciation. Nevertheless, the underlying drivers of genetic differentiation varied substantially. Synura petersenii exhibited a population pattern primarily associated with environmental factors, with conductivity emerging as the strongest habitat predictor of genetic structuring. In contrast, S. glabra demonstrated strict geographical boundaries and formed populations primarily according to geographical distance. We propose that divergences in population patterns between the two studied species arise due to differing mechanisms of distribution. By employing a balanced sampling design with comparable geographic coverage for both species, our study enables a robust comparison of their divergence and provides new insights into the complexity of speciation.

Lichens are excellent bioindicators of overall ecosystem health. The symbiotic nature of their thalli enables tracking changes in humidity, temperature, habitat disturbance and air pollution, often before larger plants do. Sensitive species usually show visible thallus damage, such as bleaching or changes in colour (including total or partial necrosis, and death of the photosynthetic component of the symbiosis), slow growth, and/or biases in reproductive strategies. Particularly, the extent to which these damages are associated with changes in the microscopic photosynthetic community inhabiting lichen thalli (phycobiota) remains poorly understood. Here, we combined Sanger and Illumina sequencing techniques to characterize the diversity and community structure of the eukaryotic phycobiome in selected epiphytic macrolichens showing different levels of thallus damage. Phylogenetic analyses revealed a high microalgal diversity, largely dominated by a few Trebouxia species, which are the most prevalent lichenized microalgae, accompanied by several low-abundance co-occurring genera. Notably, microalgal diversity peaked at intermediate levels of thallus damage. This pattern is consistent with disturbance-mediated modulation of microalgal community evenness rather than a categorical shift in symbiotic composition. These findings reveal previously unrecognized variability within the lichen phycobiota, providing new insights into the ecological dynamics and stress responses of these communities. In conclusion, our work offers a new perspective on the potential of lichens as sensitive bioindicators of air quality and ecosystem health.

Papillae are small siliceous protuberances occurring on the scales and cysts of many chrysophyte algae, yet the environmental factors shaping their distribution remain poorly understood. We combined pan-European and local datasets to identify the key drivers of papillae formation on the scales of the chrysophyte genus Mallomonas Perty. At the continental scale, warmer temperatures and low pH were associated with a higher proportion of papillae-forming species. At the local scale, low pH was again confirmed as the strongest predictor, along with elevated dissolved silica concentrations. The observed temperature effect is consistent with the idea that papillae help maintain hydrodynamic performance in warmer, less viscous water by modifying microscale flow and supporting nutrient exchange. The strong negative relationship with pH suggests that papillae may also mitigate chemical stress, for example by stabilizing the silica scale case or influencing interactions with metal ions. Together, these results show that both broad climatic gradients and fine-scale water chemistry shape the occurrence of papillae-forming species. By linking environmental conditions to variation in surface ornamentation, this study provides new insight into the ecological significance of papillae and the environmental pressures that may have driven their repeated evolution in chrysophyte algae.

Mallomonas is among the most diverse genera of Chrysophyceae, yet many of its lineages remain taxonomically unresolved due to limited molecular sampling. Mallomonas series Peronoides, historically limited to four species, has posed persistent taxonomic difficulties, most notably the long-standing and problematic reliance on a rare hemispherical structure to distinguish M. bangladeshica from M. peroneides. Here, we present a comprehensive revision of Mallomonas section Peroneides stat. nov., formerly treated as ser. Peronoides, that integrates detailed morphological analyses with a five-gene phylogeny. Thirty-one strains isolated from 15 freshwater sites across five countries resolved into seven well-supported clades based on the nuclear ribosomal internal transcribed spacer (ITS) DNA region. Four corresponded to previously described species (M. bangladeshica, M. ceylanica, M. peroneides, M. stellata), whereas three represente novel taxa: M. cotyloides sp. nov., M. neotropica sp. nov., and M. palmata sp. nov. Morphological reassessment, including original iconotypes, identified reliable diagnostic characters in scale architecture; secondary layer distribution; and experimentally confirmed, temperature-dependent formation of the hemispherical structure. Phylogenetic analyses revealed two deeply divergent lineages within the section, reflecting distinct evolutionary trajectories in central depression morphology and scale diversification. Biogeographic and ecological data indicated a primary association with warm, humic freshwater habitats, although several taxa tolerate markedly cooler conditions. Together, these results refine species boundaries, resolve long-standing taxonomic ambiguities, and establish a robust framework for future taxonomic, phylogenetic, and ecological research on Mallomonas.

Genome size varies tremendously across eukaryotes, which often contain far more DNA than expected from their biological complexity. To explain this paradox, selection-based hypotheses propose that genome size evolves through selection acting on life-history traits correlated with the phenotypic effects, independent of its genic content. To test the association between genome size and resting cyst size-a structure with morphology that may reflect overall body size-we selected chrysophyte algae (Chrysophyceae) producing siliceous stomatocysts as a model. In this study, we obtained and identified 85 chrysophyte strains representing 31 Mallomonas species using nuclear ITS rDNA region barcoding and estimated their genome sizes using propidium iodide flow cytometry. Within this genus, we observed more than a 75-fold variation in genome size (0.15-11.25 pg) and frequent substantial intraspecific variation, in some cases consistent with whole-genome doubling (polyploidization). By summarizing the published genome size records, adding our new measurements and combining them with published cyst sizes, we assembled a data set for 54 chrysophyte species to examine the relationship between genome size and cyst volume. Linear regression on log-transformed data revealed a strong positive correlation ( R adj 2  = 0.626, p < 0.001), showing that species with larger genomes tend to produce larger stomatocysts. In chrysophytes, this relationship is considerably stronger than the widely documented genome size-cell size correlation observed across eukaryotes. Our findings suggest that stomatocyst size is a more informative predictor of genome size in this group and highlight the potential for using stomatocyst sizes to infer ancestral genome sizes from the fossil record.

Genome size is a fundamental characteristic of the cell and is associated with a number of key features of the organism, such as cell size, division rate or metabolic rate. Knowledge of the genome size is also a prerequisite for many areas of research (e.g. selection of suitable organisms for whole genome sequencing or cell cycle analysis). However, genome size analysis in microalgae is often difficult and involves many methodological challenges. As a result, genome size data for microalgae are largely lacking. In this study, we focused on fragile, poorly growing colonial chrysophytes. We analysed their genome size using flow cytometry and tested the difference between colonial and solitary living chrysophytes on all published genome size data using analysis of variance. We successfully established nine cultures that were further determined to belong to six species of colonial chrysophytes. We estimated their genome size to be on average 0.24 pg & centerdot;cell-1 for Chrysosphaerella longispina Lauterborn, 1.70 pg & centerdot;cell-1 for Neotessella lapponica (Skuja) B.Y.Jo, J.I.Kim, W.Shin, P.& Scaron;kaloud & P.A.Siver, 0.25 pg & centerdot;cell-1 for Uroglenopsis turfosa (Skuja) R.H.Thompson & D.E.Wujek, 0.31 pg & centerdot;cell-1 for Urostipulosphaera articulata (Korshikov) Pusztai & & Scaron;kaloud, 0.22 pg & centerdot;cell-1 for U. granulata Pusztai & & Scaron;kaloud and 0.19 pg & centerdot;cell-1 for U. lindiae (Bourrelly) Pusztai & & Scaron;kaloud. It was also shown that colonial chrysophytes have larger genomes compared to solitary living species. This study further revealed the smallest genome among colonial chrysophytes, belonging to Urostipulosphaera lindiae. Nevertheless, colonial chrysophytes have lower variance in genome size, possibly due to evolutionary constraints on cell size (and genome size) variation to maintain the functionality of the whole colony movement.

A total of 81 strains morphologically corresponding to the circumscription of Mallomonas intermedia were isolated from 27 freshwater localities across Europe and North America. Molecular genetic analyses revealed two distinct lineages with a strict geographical pattern, which diverged approximately 8 million years ago. According to comparative morphological analyses, we concluded that European populations corresponded to M. intermedia, whereas North American populations were described as a new species, M. retimedia sp. nov. The most notable morphological difference between the species is in the number of pores enclosed in the mesh formed on the siliceous scales. While in M. intermedia the mesh typically surrounds only one pore, meshes of M. retimedia usually encircle 3-4 pores. By incorporating fossil evidence into our time-calibrated phylogeny, we propose that the ancestor of M. intermedia and six related species likely lacked a thick secondary layer but had a distinct transverse rib, present in extant M. galeiformis, M. intermedia and M. retimedia species. The evolutionary history of this group further reveals that parallel ribs on the siliceous scales have evolved independently at least three times during the Miocene epoch, suggesting an adaptive significance, although their exact function remains unclear.

Loricate golden algae (Chrysophyceae) are photosynthetic microorganisms characterized by a lorica, a rigid or semi-rigid protective casing made of organic material, sometimes reinforced with silica or iron. The lorica's diverse shapes and intricate ornamentation serve as both adaptive strategies and taxonomic markers. Here, we identified, for the first time, the molecular phylogenetic position of a loricate genus Chrysococcus, based on genetic investigations of two freshwater populations in Poland. The genus was resolved to form a wellsupported clade with Chrysosaccus within the order Chrysosaccales. Accordingly, this order represents one of the morphologically most diverse lineages of Chrysophyceae, including naked flagellates, coccoid organisms, amoebae and flagellates dwelling in loricae, and mucilage-secreting cells. The phylogenetic resolution of Chrysococcus provides key evidence for understanding the evolutionary transitions within Chrysophyceae, highlighting the complex relationships between loricate and non-loricate taxa.

We conducted a survey of Synurales diversity in Florida (United States), focusing on two established hotspots-Ocala National Forest and Myakka River State Park-and two previously unexplored sites-Manatee Lake and Lake Lee. Using transmission electron microscopy (TEM), we identified 69 species, increasing the total number recorded from Florida to 90. Among these, three species-Mallomonas cornea sp. nov., M. laureana sp. nov., and M. joergenii sp. nov.-have been newly described based on detailed morphological and molecular analyses. We have also proposed a new combination, M. poseidonii comb. et stat. nov., and established a new section, Corneae sect. nov., based on molecular phylogenetics. Our findings revealed an independent emergence of ribbed scale shield ornamentation in M. joergenii sp. nov. and suggested that internal scale reticulation in M. laureana sp. nov. may be an adaptation to high UV irradiance in tropical environments. This study underscores the value of DNA sequence data in resolving taxonomic ambiguities, elucidating evolutionary patterns, and enhancing species recovery from type localities.

Synura represents a common freshwater protist genus known for producing morphologically diverse silica scales with species-specific secondary structures. In this study, we analysed 19 scale-bearing morphological traits across over 700 genetically verified records, spanning a broad environmental gradient in Europe. Focusing on Synura from section Petersenianae, we identified 29 species-level lineages, 13 of which had not been previously described. Interestingly, while species diversity increased northwards, morphological trait diversity exhibited the opposite trend, being higher in southern Europe. This suggests that abiotic factors play a significant role in shaping these traits, which are differently responsive to gradients than taxonomic diversity. We observed non-random distribution of scale morphological traits, influenced by abiotic factors such as temperature, precipitation and pH. Scale elongation appears to be an adaptation for living in oligotrophic waters with reduced sunlight availability, while the increasing distance between struts likely reflects an adaptation to low silica concentrations. Notably, specific morphological traits may provide more informative insights into environmental drivers than taxonomic units. The study sheds light on the intricate relationship between environmental conditions and the morphology of silica scales, emphasizing the importance of considering both taxonomic and morphological diversity in ecological research.

Symbiotic systems of photosynthetic microorganisms and fungi are widespread in terrestrial biomes and lichens are probably the most advanced and complex. Conversely, the least complex systems are "green biofilms" with a completely unexplored mycobiome. We describe here a new system intermediate between green biofilms and lichens - semilichens. Light and fluorescence microscopy, eDNA sequencing, molecular phylogeny, Chlorophyll a fluorescence and 13C labelling/metabolomics were used to study algal and fungal identity, morphology and physiology of the symbiosis. Tight contact between algae and a single predominant fungus (mycobiont) is revealed in semilichens. The algae are from the symbiotic lineages of Trebouxiophyceae and Ulvophyceae, the fungi belong to Arthoniomycetes, Dothideomycetes, Eurotiomycetes, Lecanoromycetes and Lichinomycetes. Algae are alive and perform substantial photosynthetic activity. 13C labelled photosynthates are partially converted into specific fungal polyols (arabitol, mannitol) demonstrating the C-flow from algae to fungi. The new symbiotic system was defined and compared with other terrestrial algal-fungal symbioses. It is characterized by minimalist environmental requirements and extremely low production of biomass. As a result, it also inhabits environments unfavourable for lichens. Our research supports the hypothesis that the long-term existence of algae and fungi in terrestrial conditions affected by frequent and repeated drying is likely dependent on their mutual coexistence.

Phytoplankton, as primary producers, play a key role in aquatic ecosystems. Their community turnover is shaped by morphological traits that enable adaptation to diverse abiotic and biotic factors. Yet, the temporal scale of these dynamics remains poorly understood due to limited high-frequency sampling studies. Employing DNA metabarcoding, we assessed the community composition of the phytoplankton lineage Synurales (Chrysophyceae) at 3-d intervals during 70 d at a shallow peat bog lake in the Czech Republic. The selected group possesses a variety of species-specific key morphological traits, such as cell size, coloniality, and bristle formation. Using a custom reference database of cultured species, we assigned 99.93% of eDNA reads to 74 species-level lineages with known morphological traits. Community changes in colonial species were influenced by abiotic drivers such as silica concentration and wind speed. By contrast, shifts in unicellular species communities were mainly driven by Cladocera predators, influencing the occurrence of bristle-bearing species. Changes in species composition and morphological traits occurred within days, mirroring environmental variability. Achieving such fine-scale resolution, especially for small or rare taxa, would be extremely difficult using microscopy alone. eDNA enabled high-resolution community profiling and abundance estimation, demonstrating its key role and the importance of comprehensive reference databases.

In mutualistic systems, the ability to associate with diverse symbionts of distinct physiological traits often facilitates broadening of the niche. In lichen symbiosis, this process remains understudied. While such flexibility has been demonstrated for some species, others associate only with symbionts of comparable characteristics. We investigated whether the broad niche of Hydropunctaria-a genus of seashore lichens inhabiting the supralittoral zone across a salinity gradient-is linked to algal symbiont turnover in response to the changing salinity. Using Sanger sequencing of lichen symbionts and Illumina metabarcoding of free-living algae, we assessed symbiont diversity, selectivity, and the presence of algal symbionts in the environment. Despite the presence of multiple potential algal partners in the surrounding environment, Hydropunctaria exhibited a highly specific and stable association with a single algal symbiont across a wide salinity range. This suggests that the ecological niche breadth in this symbiotic system is driven not by the ability to change partners in response to changing salinity, but rather by the association between generalist, euryhaline symbionts. Our findings also point to selective mechanisms beyond partner availability alone.

Understanding the initial formation and development of lichens is crucial for elucidating the mechanisms behind the formation of complex lichen thalli and their maintenance in long-term symbioses. These symbiotic relationships provide significant ecological advantages for both partners, expanding their ecological niches and allowing them, in many cases, to overcome extreme environmental conditions. The correct development of thalli likely relies on the selection of suitable photobionts from the environment. In this study, we focused on the impact of lichen age on the overall diversity of photobiont partners and examined how mycobiont preference toward their symbionts changes at different developmental stages. Using the lichen Protoparmeliopsis muralis as a model organism, we observed a strong correlation between the diversity of photobionts and lichen age, confirmed by both molecular data and morphological observations. Our findings indicate greater photobiont diversity in older thalli, suggesting that lichens retain the majority of algae they collect throughout their lifespan, potentially as an adaptation to changing environmental conditions. Additionally, we found that some lichen samples contained only low levels of Trebouxia algae, indicating that P. muralis does not consistently rely on this typical partner and that local environmental conditions may significantly influence its symbiotic composition.

The nuclear genome is essential for encoding most of the genes required for cellular processes, but its size alone can alter the characteristics of cells and organisms. Yet, genome size variation and its ecological and evolutionary impacts, particularly in microorganisms, are not well understood. We used flow cytometry to estimate genome size and GC content in 53 evolutionary lineages of the microalgal genus Synura (Chrysophyceae, Stramenopiles). Genome size evolution was reconstructed in a phylogenetic framework using molecular markers. A set of genomic, morphological, and ecogeographic variables characterizing Synura lineages was evaluated and tested as predictors of genome size variation in phylogeny-corrected statistical models. Both genome size and GC content varied widely in Synura, ranging from 0.19 to 3.70 pg of DNA and 34.0% to 49.3%, respectively. Genome size variation was mainly associated with cell size, less with silica scale size, and not with scale ultrastructure. Higher soil nitrogen, higher latitudes, and lower temperatures correlated with larger genomes. Genome size evolution in Synura shows potential dynamism, with increases confined to short terminal branches, indicating lower macroevolutionary stability. Lineages with larger genomes exhibited a narrower range of suitable ecological conditions, possibly due to selection acting deleteriously against larger genomes (and cells).

We present a synopsis of studies reporting the free-living occurrence of green algal lichen symbionts. We give an overview of all known lichen photobiont genera together with comprehensive descriptions, taxonomical classification and occurrence data. Based on the analysis of 310 records, we discovered that at least 80% of lichen photobiont genera were observed in the free-living state. Diplosphaera chodatii, Elliptochloris bilobata and Chloroidium ellipsoideum represent both morphologically and genetically the most frequently reported free-living photobiont species. Trebouxia, the most prevalent genus of lichen photobionts, has frequently been reported to exist independently to fungal hyphae. Based on our literature survey, free-living photobionts of lichens are able to grow in a wide range of environments and substrates, with most records coming from soil and biological soil crusts.

Chrysophytes are a diverse group of stramenopile protists comprising a variety of taxa with different cell organizations. Here, we morphologically and genetically characterize a novel chrysophyte genus isolated from three freshwater localities in the Czech Republic, described as Globulochrysis compacta gen. nov. sp. nov. The genus is well characterized by the frequent formation of closely packed cells, developing into large, spherical sporangia. This alga also forms solitary cells, mucilaginous colonies, amoebae, and teardrop-shaped zoids. We also investigated the morphologically similar genus Chrysotilos, isolated from its type locality in the Austrian Alps. In culture, Chrysotilos showed characteristic neustonic growth, which was, however, not observed in Globulochrysis. Phylogenetic analyses based on 18S rRNA and rbcL genes showed that these two algae belong to two distinct lineages. Whereas Chrysotilos was inferred within the clade Hibberdiales, Globulochrysis formed a completely new lineage within the order Hydrurales.

Molecular identification of micro- and macroorganisms based on nuclear markers has revolutionized our understanding of their taxonomy, phylogeny and ecology. Today, research on the diversity of eukaryotes in global ecosystems heavily relies on nuclear ribosomal RNA (rRNA) markers. Here, we present the research community-curated reference database EUKARYOME for nuclear ribosomal 18S rRNA, internal transcribed spacer (ITS) and 28S rRNA markers for all eukaryotes, including metazoans (animals), protists, fungi and plants. It is particularly useful for the identification of arbuscular mycorrhizal fungi as it bridges the four commonly used molecular markers-ITS1, ITS2, 18S V4-V5 and 28S D1-D2 subregions. The key benefits of this database over other annotated reference sequence databases are that it is not restricted to certain taxonomic groups and it includes all rRNA markers. EUKARYOME also offers a number of reference long-read sequences that are derived from (meta)genomic and (meta)barcoding-a unique feature that can be used for taxonomic identification and chimera control of third-generation, long-read, high-throughput sequencing data. Taxonomic assignments of rRNA genes in the database are verified based on phylogenetic approaches. The reference datasets are available in multiple formats from the project homepage, http://www.eukaryome.org.