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To decipher the dispersal range of the cosmopolitan green microalga Tetracystis vinatzeri, two airborne-collected strains were barcoded using the 18S ribosomal DNA gene (18S) and the mitochondrial cytochrome c oxidase I subunit (COI), and their survival capacities were investigated across a stepwise acidic to alkaline pH gradient. Genetic fingerprinting confirmed the identity of the strains as representing the same biological species. Investigations of the tolerance of T. vinatzeri to pH levels refined the delimitation of its ecological niche. T. vinatzeri showed optimal growth in freshwater habitats at pH 4.3–9.6 and strains could cope with exposure to slightly more acidic (down to 3.8) and alkaline (up to 10.4) conditions. However, organismal survival was drastically affected by exposure to pH < 3 and > 11. Morphological observations showed that T. vinatzeri could better cope with exposure to acidic pH, exhibiting less cell membrane damage. Genetic analysis showed that the two airborne strains shared the same haplotype, suggesting that they either originated from the same air mass or that the haplotype has a widespread distribution. Further phylogeographic and physiological investigations are required to define the spreading dynamic of T. vinatzeri. Highlights: Tetracystis vinatzeri can grow at pH from 3.8–10.4. Strains from different habitats showed sexual compatibility. Low genetic diversity and cosmopolitan distribution suggest widespread dispersal. [ABSTRACT FROM AUTHOR]

Limnomonas gaiensis is a recently described green microalga inhabiting freshwater lakes in northern Europe (England, Sweden). There is little information on the species' tolerance to environmental factors and its capacity to spread to unconnected lakes. Using a barcoding approach, we investigated the phylogeography of L. gaiensis along a latitudinal gradient spanning 54–60°N. Experimental long-term exposure to a gradient of water temperatures, mimicking natural conditions, was performed to assess its thermal tolerance. Genetic analyses showed that this limnic species is easily identified at the species level. Phylogeographic analyses revealed the existence of two genetically similar haplotypes over the mitochondrial cytochrome C oxidase subunit 1 gene in different countries, suggesting that the range of L. gaiensis has recently expanded from a common source lineage, despite geographic isolation. Heterogeneity in the Helix III of the nuclear Internal Transcribed Spacer 2 region of the English strain suggested past hybridization. Physiological analyses showed that L. gaiensis can survive and reproduce asexually, potentially year-round. Optimal growth was observed at 15°C, with a doubling time of 1.1 days. The species is able to survive short periods of desiccation. The lack of connectivity between the lakes where the species was sampled rules out aquatic dispersal of this species. However, its physiological features make L. gaiensis a good candidate for propagule dispersal by either wind or human/animal-mediated transportation. Our study calls for further investigation into limnic systems, using rapid barcoding, with concurrent atmospheric and physiological analyses to establish its distribution over wider geographic scales and to understand how L. gaiensis disperses. Limnomonas gaiensis survives in a wide range of water temperatures. Phylogeographic analyses indicate its recent expansion in Northern Europe. Physiological features suggest airborne/animal-mediated dispersal between unconnected freshwater lakes. [ABSTRACT FROM AUTHOR]

Background. Zooplankton plays an important role in the marine ecosystem. A high level of taxonomic expertise is necessary for accurate species identification based on morphological characteristics. As an alternative method to morphological classification, we focused on a molecular approach using 18S and 28S ribosomal RNA (rRNA) gene sequences. This study investigates how the accuracy of species identification by metabarcoding improves when taxonomically verified sequences of dominant zooplankton species are added to the public database. The improvement was tested by using natural zooplankton samples. Methods. rRNA gene sequences were obtained from dominant zooplankton species from six sea areas around Japan and registered in the public database for improving the accuracy of taxonomic classifications. Two reference databases with and without newly registered sequences were created. Comparison of detected OTUs associated with single species between the two references was done using field-collected zooplankton samples from the Sea of Okhotsk for metabarcoding analysis to verify whether or not the newly registered sequences improved the accuracy of taxonomic classifications. Results. A total of 166 sequences in 96 species based on the 18S marker and 165 sequences in 95 species based on the 28S marker belonging to Arthropoda (mostly Copepoda) and Chaetognatha were registered in the public database. The newly registered sequences were mainly composed of small non-calanoid copepods, such as species belonging to Oithona and Oncaea. Based on the metabarcoding analysis of field samples, a total of 18 out of 92 OTUs were identified at the species level based on newly registered sequences in the data obtained by the 18S marker. Based on the 28S marker, 42 out of 89 OTUs were classified at the species level based on taxonomically verified sequences. Thanks to the newly registered sequences, the number of OTUs associated with a single species based on the 18S marker increased by 16% in total and by 10% per sample. Based on the 28S marker, the number of OTUs associated with a single species increased by 39% in total and by 15% per sample. The improved accuracy of species identification was confirmed by comparing different sequences obtained from the same species. The newly registered sequences had higher similarity values (mean >0.003) than the pre-existing sequences based on both rRNA genes. These OTUs were identified at the species level based on sequences not only present in the Sea of Okhotsk but also in other areas. Discussion. The results of the registration of new taxonomically verified sequences and the subsequent comparison of databases based on metabarcoding data of natural zooplankton samples clearly showed an increase in accuracy in species identification. Continuous registration of sequence data covering various environmental conditions is necessary for further improvement of metabarcoding analysis of zooplankton for monitoring marine ecosystems. [ABSTRACT FROM AUTHOR]

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., (© The Author(s) 2024. Published by Oxford University Press.)