Genomes of Subaerial Zygnematophyceae Provide Insights into Land Plant Evolution.

The transition to a terrestrial environment, termed terrestrialization, is generally regarded as a pivotal event in the evolution and diversification of the land plant flora that changed the surface of our planet. Through phylogenomic studies, a group of streptophyte algae, the Zygnematophyceae, have recently been recognized as the likely sister group to land plants (embryophytes). Here, we report genome sequences and analyses of two early diverging Zygnematophyceae (Spirogloea muscicola gen. nov. and Mesotaenium endlicherianum) that share the same subaerial/terrestrial habitat with the earliest-diverging embryophytes, the bryophytes. We provide evidence that genes (i.e., GRAS and PYR/PYL/RCAR) that increase resistance to biotic and abiotic stresses in land plants, in particular desiccation, originated or expanded in the common ancestor of Zygnematophyceae and embryophytes, and were gained by horizontal gene transfer (HGT) from soil bacteria. These two Zygnematophyceae genomes represent a cornerstone for future studies to understand the underlying molecular mechanism and process of plant terrestrialization.

Immunomodulatory Effect of Flammulina rossica Fermentation Extract on Healthy and Immunosuppressed Mice.

Flammulina rossica fermentation extract (FREP) was obtained by ethanol precipitation of the fermentation broth. The molecular weight of FREP is 28.52 kDa, and it mainly contains active ingredients such as polysaccharides, proteins, reducing sugars, and 16 amino acids. Among them, the polysaccharides were mannose, glucose, galactose, arabinose, and fucose and possessed ß-glycosidic bonds. Furthermore, the immunoregulatory activities of FREP were investigated in vivo. The results demonstrated that FREP could increase the counts of CD4+ T lymphocytes and the ratio of CD4+/CD8+ in a dose-dependent manner in healthy mice. In addition, FREP significantly increased serum cytokines, including IL-2, IL-8, IL-10, IL-12, IL-6, IL-1ß, INF-γ, C-rection protein, and TNF-α, and promoted splenocyte proliferation in healthy mice. Finally, FREP could restore the counts of white blood cells, red blood cells, secretory immunoglobulin A, and antibody-forming cells and significantly promote the serum haemolysin level in mice treated with cyclophosphamide. The findings indicated that FREP possessed immunoregulatory activity in healthy mice and could improve the immune functions in immunosuppressive mice. Therefore, FREP could be exploited as an immunomodulatory agent and potential immunotherapeutic medicine for patients with inadequate immune function.

Diversity of bacterial communities in the plasmodia of myxomycetes.

BACKGROUND: Myxomycetes are a group of eukaryotes belonging to Amoebozoa, which are characterized by a distinctive life cycle, including the plasmodium stage and fruit body stage. Plasmodia are all found to be associated with bacteria. However, the information about bacteria diversity and composition in different plasmodia was limited. Therefore, this study aimed to investigate the bacterial diversity of plasmodia from different myxomycetes species and reveal the potential function of plasmodia-associated bacterial communities. RESULTS: The bacterial communities associated with the plasmodia of six myxomycetes (Didymium iridis, Didymium squamulosum, Diderma hemisphaericum, Lepidoderma tigrinum, Fuligo leviderma, and Physarum melleum) were identified by 16S rRNA amplicon sequencing. The six plasmodia harbored 38 to 52 bacterial operational taxonomic units (OTUs) that belonged to 7 phyla, 16 classes, 23 orders, 40 families, and 53 genera. The dominant phyla were Bacteroidetes, Firmicutes, and Proteobacteria. Most OTUs were shared among the six myxomycetes, while unique bacteria in each species only accounted for a tiny proportion of the total OTUs. CONCLUSIONS: Although each of the six myxomycetes plasmodia had different bacterial community compositions, a high similarity was observed in the plasmodia-associated bacterial communities' functional composition. The high enrichment for gram-negative (> 90%) and aerobic (> 99%) bacteria in plasmodia suggest that myxomycetes may positively recruit certain kinds of bacteria from the surrounding environment.

Preliminary Analysis of Phylogenetic Relationships of the Asian-Pacific Endemial Subterranean Amphipod Genus Pseudocrangonyx Among Families and Genera of Crangonyctoidean Amphipods Inferred by Partial LSU rDNA Gene Sequences.

To analyze the phylogenetic relationships of the genus Pseudocrangonyx within the superfamily Crangonyctoidea, we sequenced a partial LSU rDNA gene from four species (six specimens). The monophyly of Pseudocrangonyx and its affinity to Crymostygius were strongly supported by four reconstruction methods: neighbor-joining, minimum evolution, maximum-likelihood, and Bayesian inference. Current geographical distributions of the taxa studied and their phylogenetic relationships established in our study suggest that members of Pseudocrangonyctidae and Crymostygius diverged from a common Crangonyx-like ancestor in the Arctic Basin region of the northern hemisphere.

Study on Syntaxonomic Diversity of Algal Cenoses in Soils of the Russian Far East, Using an Integrative Taxonomic Approach.

Soil is a unique ecosystem with peculiar biodiversity that includes cyanobacteria and algae. Traditionally, cyanobacterial and algal cenoses were described mainly using the dominance approach, rarely based on the Braun-Blanquet method (floristic classification). More importantly, in both cases, the species of cyanobacteria and algae in communities were identified using classical methods (light microscopy) only. In this study, we present results of soil algal cenoses classification using the Braun-Blanquet approach based on species composition data obtained via an integrative approach. Characteristic tables include 19 out of 108 samples collected in the Jewish Autonomous Region, Primorsky Territory, and Sakhalin Region (Iturup Island) in 2018 and in 2020-2021. Twenty-five species of algae from four classes were identified in these sites. We described three new associations of algal communities-Coelastrelletum aeroterrestricae ass. nova, Vischerietum magnae ass. nova, Bracteacoccetum bullati ass. nova. PCA analysis corroborated the results of syntaxonomic analysis and revealed that Coelastrelletum aeroterrestricae inhabit soils with a high value of P; Vischerietum magnae inhabit soils with high value of soil organic carbon (SOC), N, and higher humidity; and Bracteacoccetum bullati inhabit soils with high K values.

Studies on subterranean amphipod crustaceans of Primory, Russia. Part 1. Three new species of the genus Pseudocrangonyx from springs and other groundwater habitats in far eastern Russia.

Three new species of the subterranean amphipod of the genus Pseudocrangonyx: P. tiunovi sp. nov., P. holsingeri sp. nov., P. sympatricus sp. nov. and male specimen for P. kseniae Sidorov, 2012 from the South Primory, Far East of Russia are described. The species were found in springs and related groundwater habitats of several river systems belonging to the Japan Sea basin. Phylogenetic analysis based on mt-cox1 sequences supported the species distinctness. A key for the South Primory species of Pseudocrangonyx is provided.

Oogamochlamys kurilensis sp. nov. (Chlorophyta, Volvocales) from the Soils of Iturup Island (Sakhalin Region, Russia).

A strain of oogamous biflagellate green alga was isolated during a study on soil algal diversity in the Russian Far East (Sakhalin Region, Iturup Island) and examined using an integrative approach. Phylogenetic analyses, based on the SSU rDNA gene, resolved the new strain as a part of the RL clade (sensu Watanabe and Nakada) within Oogamochlamydinia (Volvocales, Chlorophyceae). The strain was similar to members of the genus Oogamochlamys (parietal and massive cup-shaped chloroplasts; two apical contractile vacuoles), but was, however, distinguished from them based on the size and shape of the mature vegetative cells, the flagellar length, the presence of only one pyrenoid in both the mature vegetative cells and the zoospores, the anterior nucleus position, and the spermatozoids' shape. Although a concept of the genus Oogamochlamys has been compromised in recent phylogenetic analysis based on the SSU rDNA sequence data and its likely affinity to anisogamous Chlamydomonas allensworthii, we described the strain from Iturup Island as Oogamochlamys kurilensis sp. nov.

Luticola tenera sp. nov. (Diadesmidaceae, Naviculales)-A New Diatom from the Soil of the State Nature Reserve "Bastak" (Jewish Autonomous Region, Russia).

Diatoms inhabit different aquatic and non-aquatic environments, including soils. The naviculoid genus Luticola is widespread in various habitats and accounts for 264 species that are only based on morphological and morphometric characteristics. These parameters can greatly vary during the life cycle, making the species very similar to each other and complicating their unambiguous identification. During a study on soil algal diversity in the Russian Far East (Jewish Autonomous Region), we isolated a strain of naviculoid diatom and examined it using an integrative approach (phylogenetic, morphological, ultrastructural data, and life cycle). Phylogenetic analyses, based on chloroplast rbcL gene data, showed affinity of the new strain with the genus Luticola. Our alga shares morphological features typical of the genus members but differs from them by having valves with a larger width and hook-shaped external proximal raphe ends deflected to the side opposite the stigma. It was revealed that the strain reproduces via two types of sexual reproduction-isogamy and cis-anisogamy. Based on these phenotypic traits, we described the new isolate as Luticola tenera sp. nov.

Ulosarcina terrestrica gen. nov., sp. nov., a New Ulvophycean Sarcinoid Alga from the Russian Far East.

Many filamentous and sarcinoid terrestrial or freshwater green algae that were previously assigned to the Chlorophyceae are members of lineages belonging to the class Ulvophyceae. One of these lineages is the Planophila-clade (Ulotrichales). Some genera in this clade share similar morphology: cell packages forming branched pseudofilaments, uniseriate or sometimes biseriate filaments, often embedded in common mucilage. During a study on soil algal diversity in the temperate monsoon climate zone in Russia (Primorsky Territory, Vladivostok), we isolated a strain of sarcinoid green alga and examined it using an integrative approach. SSU and ITS rDNA sequence data, morphological characteristics, and life cycle features differentiated this strain from closely related genera of the order Ulotrichales and led us to describe it as Ulosarcina terrestrica gen. et sp. nov.

Structural characterisation and antitumor activity against non-small cell lung cancer of polysaccharides from Sanghuangporus vaninii.

The medicinal fungus Sanghuangporus vaninii can be cultivated in large scale and has outstanding antitumour activity. In this study, water-soluble S. vaninii polysaccharides (SVPs) were extracted from fruiting bodies. Four polysaccharide sub-fractions (SVP-W, SVP-1, SVP-2 and SVP-3) were isolated, with molecular weights from 90.50 kDa to 261.70 kDa, and all inhibited the proliferation of non-small cell lung cancer cell lines A549, 95-D and NCI-H460, especially the acidic SVP-1. SVP-1 affected cell morphology and colony formation in NCI-H460 cells. It also promoted cell apoptosis following nuclear fluorescence staining and flow cytometry. Methylation and nuclear magnetic resonance analyses revealed that SVP-1 is a heteroglycan with the main chain →4)-ß-D-Glcp-(1 â†’ 6)-ß-D-Glcp-(1 â†’ 6)-α-D-Galp-(1 â†’ 6)-ß-D-Glcp-(1→, and the branched chain α-D-Manp-(1 â†’ 2)-α-D-Manp-(1 â†’ 3)-ß-D-Glcp-(1 â†’ 3,6)-ß-D-Glcp-(1→. The findings indicate that this natural acidic polysaccharide has potential for non-small cell lung cancer therapy.

New Species of Chloroidium (Trebouxiophyceae, Chlorophyta) from East Asia.

Chlorella-like green algae that reproduce only asexually by immotile autospores or motile zoospores are the most typical inhabitants of non-aquatic environments. They have a simple morphology that hampers their differentiation, but algae of such habit represent a diverse array of lineages, which are mostly in the classes Chlorophyceae and Trebouxiophyceae. One of these lineages is the order Watanabeales (Watanabea clade; Trebouxiophyceae), which comprises 10 genera that share a distinct mode of reproduction through unequally sized autospores. Most of these genera account for a few species that are rarely recorded in nature. In contrast, the genus Chloroidium is one of the most species-rich and widely distributed members of the order. Three strains of coccoid green alga were isolated during a study of soil algae in the temperate monsoon climate zone of Asia. These strains are described here as a new species, Chloroidium orientalis. SSU and ITS rDNA sequence data, morphological characteristics, and life cycle features differentiate these strains from known members of the genus.

Integrative Taxonomic, Ecological and Genotyping Study of Charophyte Populations from the Egyptian Western-Desert Oases and Sinai Peninsula.

Present-day information available on the charophyte macroalgae in Egypt, including their phylogenetic affinities, remains largely incomplete. In this study, nine charophyte populations were collected from different aquatic biotopes across the Egyptian Western-Desert Oases and Sinai Peninsula. All populations were investigated using an integrative polyphasic approach including phylogenetic analyses inferred from the chloroplast-encoded gene (rbcL) and the internal transcribed spacer (ITS1) regions, in parallel with morphotaxonomic assignment, ultrastructure of the oospore walls, and autecology. The specimens identified belonged to the genera Chara, Nitella, and Tolypella, with predominance of the first genus to which five species were assigned though they presented some interesting aberrant taxonomic features: C. aspera, C. contraria, C. globata, C. tomentosa, and C. vulgaris. Based on our integrative study, the globally rare species C. globata was reported for the second time for the whole African continent. The genus Nitella was only represented by N. flagellifera, and based on the available literature, it is a new record for North Africa. Noteworthy, an interesting Tolypella sp., morphologically very similar to T. glomerata, was collected and characterized and finally designated with the working name 'Tolypella sp. PBA-1704 from a desert, freshwater wetland', mainly based on its concatenated rbcL+ITS1 phylogenetic position. This study not only improved our understanding on the diversity, biogeography and autecological preferences of charophytes in Egypt, but it also broadened our knowledge on this vulnerable algal group in North Africa, emphasizing the need of more in-depth research work in the future, particularly in the less-impacted desert habitats.

Characterization of a polysaccharide from Sanghuangporus vaninii and its antitumor regulation via activation of the p53 signaling pathway in breast cancer MCF-7 cells.

Sanghuangporus is a traditional medicine that has been used for nearly 2000 years in Asia. It has been found to enhance immunity and have anticancer properties. Some studies determined that Sanghuangporus polysaccharide can inhibit tumors in vitro, although the underlying mechanisms remain unknown. Therefore, the present study investigated the antitumor effects of polysaccharides from the fruiting body of S. vaninii and their associated mechanisms. Water-soluble S. vaninii polysaccharide (SVP) with a molecular weight of 3.156 × 104 Da was isolated and found to be mainly composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucosamine, glucose, galactosamine, galactose, xylose, arabinose, and fucose in molar ratios of 1.63:0.04:0.36:0.03:0.13:8.39:0.08:1.08:0.25:1.07:0.40. SVP showed modest anti-proliferative activity against HeLa, SHG-44, SMMC-7721, and MCF-7 cells. Furthermore, it effectively regulated cell cycle, promoted apoptosis, and reduced the migratory and invasive capacities of MCF-7 cells. Mechanistically, SVP enhanced activation of p53-related genes and down-regulated MMP expression. These findings describe a potential natural polysaccharide for tumor therapy and a basis for the development of fungal polysaccharides as a type of health food.

Dictyostelid Cellular Slime Molds from the Russian Far East.

Dictyostelid cellular slime molds (dictyostelids) are important soil microorganisms that feed mostly on bacteria in the soil and leaf litter layer. The Russian Far East is the easternmost part of Russia and thus is located in the Middle East of Siberia. In September 2018, 14 samples of mixed soil/humus were collected from the most southeastern portion of the Russian Far East, including Sakhalin Island, Ussuriysk and Vladivostok, and then processed for dictyostelids. Seven species in four genera were recovered. Four of these species (Cavenderia fasciculata, Heterostelium tenuissimum, Dictyostelium longosporum and Polysphondylium patagonicum) were recorded for the first time from Russia, and one species (H. multibrachiatum sp. nov.) in described herein as new to science.

A study of conflict between molecular phylogeny and taxonomy in the Desmidiaceae (Streptophyta, Viridiplantae): analyses of 291 rbcL sequences.

Molecular phylogenetic analyses of 93 new and 198 non-redundant GenBank rbcL sequences of the family Desmidiaceae (Zygnematophyceae, Streptophyta) established 22 mostly highly supported clades, in addition to four non-supported lineages and eight single-taxon branches within the family. Nine novel clades and single-taxon branches were identified, suggesting that current taxon sampling has not reached saturation in the family. The highly polyphyletic nature of most desmid genera corroborated in this study using a large taxon set, calls for re-evaluation of the genus concept in the family Desmidiaceae that traditionally relied on features of cell morphology. Molecular phylogenetic data have shown that these morphological characters are highly homoplastic or plesiomorphic and thus cannot be used to delineate genera. The dramatic discrepancy between the currently practised systematic treatment of the family and the composition of the clades based on sequence comparisons requires emendation of almost all existing genera and description of a larger number of novel genera. The clades identified during this study provide a framework for the future emendation/description of genera in the Desmidiaceae.

In search of monophyletic taxa in the family Desmidiaceae (Zygnematophyceae, Viridiplantae): the genus Cosmarium.

Nuclear-encoded small subunit (SSU) rDNA, 1506 group I introns, and chloroplast rbcL genes were sequenced from 97 strains representing the largest desmid genus Cosmarium (45 spp.), its putative relatives Actinotaenium (5 spp.), Xanthidium (4 spp.), Euastrum (9 spp.), Staurodesmus (13 spp.), and other Desmidiaceae (Zygnematophyceae, Streptophyta) and used to assess phylogenetic relationships in the family. Analyses of single genes and of a concatenated data set (3260 nt) established 10 well-supported clades in the family with Cosmarium species distributed in six clades and one nonsupported assemblage. Most of the clades contained representatives of at least two genera highlighting the polyphyletic nature of the genera Cosmarium, Euastrum, Staurodesmus, and Actinotaenium. To enhance resolution between clades, we extended the data set by sequencing the slowly evolving chloroplast-encoded large subunit (LSU) rRNA gene from 40 taxa. Phylogenetic analyses of a concatenated data set (5509 nt) suggested a sister relationship between two clades that consisted mainly of Cosmarium species and included C. undulatum, the type species of the genus. We describe molecular signatures in the SSU rRNA for two clades and conclude that more studies involving new isolates, additional molecular markers, and reanalyses of morphological traits are necessary before the taxonomic revision of the genus Cosmarium can be attempted.

Ribosomal ITS sequences allow resolution of freshwater sponge phylogeny with alignments guided by secondary structure prediction.

Freshwater sponges include six extant families which belong to the suborder Spongillina (Porifera). The taxonomy of freshwater sponges is problematic and their phylogeny and evolution are not well understood. Sequences of the ribosomal internal transcribed spacers (ITS1 and ITS2) of 11 species from the family Lubomirskiidae, 13 species from the family Spongillidae, and 1 species from the family Potamolepidae were obtained to study the phylogenetic relationships between endemic and cosmopolitan freshwater sponges and the evolution of sponges in Lake Baikal. The present study is the first one where ITS1 sequences were successfully aligned using verified secondary structure models and, in combination with ITS2, used to infer relationships between the freshwater sponges. Phylogenetic trees inferred using maximum likelihood, neighbor-joining, and parsimony methods and Bayesian inference revealed that the endemic family Lubomirskiidae was monophyletic. Our results do not support the monophyly of Spongillidae because Lubomirskiidae formed a robust clade with E. muelleri, and Trochospongilla latouchiana formed a robust clade with the outgroup Echinospongilla brichardi (Potamolepidae). Within the cosmopolitan family Spongillidae the genera Radiospongilla and Eunapius were found to be monophyletic, while Ephydatia muelleri was basal to the family Lubomirskiidae. The genetic distances between Lubomirskiidae species being much lower than those between Spongillidae species are indicative of their relatively recent radiation from a common ancestor. These results indicated that rDNA spacers sequences can be useful in the study of phylogenetic relationships of and the identification of species of freshwater sponges.

Agrobacterium-mediated transformation of sea urchin embryos.

Agrobacterium-mediated transformation of higher plants is a well-known and powerful tool for transgene delivery to plant cells. In the present work, we studied whether Agrobacterium can transfer genetic information to animal (sea urchin) embryos. Sea urchin embryos were co-cultivated with A. tumefaciens strains carrying binary vectors containing the nptII marker gene and agrobacterial rolC and rolB oncogenes. Bacterial plasmid T-DNA-sea urchin DNA junction sites were identified in the genome of these embryos, thus indicating successful transformation. The nptII and both rol genes were expressed in the transformed embryos. The processes of transgene integration and transgene expression were suppressed when Agrobacteria contained mutated virA, virB or virG genes, suggesting that Agrobacterium transforms sea urchin cells by a mechanism similar to that which mediates T-DNA transfer to plants. Some of the embryos co-cultivated with Agrobacterium developed teratoma-like structures. The ability of Agrobacterium strains to trigger formation of teratoma-like structures was diminished when they contained the mutated vir genes. In summary, our results demonstrate that Agrobacterium is able to transform animal (sea urchin) embryonic cells, thus indicating a potential of this natural system for gene delivery to animal hosts. We also discuss the possibility of horizontal gene transfer from Agrobacterium to marine invertebrates.

Molecular phylogeny of conjugating green algae (Zygnemophyceae, Streptophyta) inferred from SSU rDNA sequence comparisons.

Nuclear-encoded SSU rDNA sequences have been obtained from 64 strains of conjugating green algae (Zygnemophyceae, Streptophyta, Viridiplantae). Molecular phylogenetic analyses of 90 SSU rDNA sequences of Viridiplantae (inciuding 78 from the Zygnemophyceae) were performed using complex evolutionary models and maximum likelihood, distance, and maximum parsimony methods. The significance of the results was tested by bootstrap analyses, deletion of long-branch taxa, relative rate tests, and Kishino-Hasegawa tests with user-defined trees. All results support the monophyly of the class Zygnemophyceae and of the order Desmidiales. The second order, Zygnematales, forms a series of early-branching clades in paraphyletic succession, with the two traditional families Mesotaeniaceae and Zygnemataceae not recovered as lineages. Instead, a long-branch Spirogyra/Sirogonium clade and the later-diverging Netrium and Roya clades represent independent clades. Within the order Desmidiales, the families Gonatozygaceae and Closteriaceae are monophyletic, whereas the Peniaceae (represented only by Penium margaritaceum) and the Desmidiaceae represent a single weakly supported lineage. Within the Desmidiaceae short internal branches and varying rates of sequence evolution among taxa reduce the phylogenetic resolution significantly. The SSU rDNA-based phylogeny is largely congruent with a published analysis of the rbcL phylogeny of the Zygnemophyceae (McCourt et al. 2000) and is also in general agreement with classification schemes based on cell wall ultrastructure. The extended taxon sampling at the subgenus level provides solid evidence that many genera in the Zygnemophyceae are not monophyletic and that the genus concept in the group needs to be revised.

Are combined analyses better than single gene phylogenies? A case study using SSU rDNA and rbcL sequence comparisons in the Zygnematophyceae (Streptophyta).

Although the combination of different genes in phylogenetic analyses is a promising approach, the methodology is not well established and analyses often suffer from inadequate, noncongruent taxon sampling, long-branch attraction, or conflicting evolutionary models of the genes analyzed. Conflicts or congruence between multigene and single-gene phylogenies, as well as the assumed superiority of the multigene approach, are often difficult to assess solely because of incongruent taxon sampling. In the present study, a data set of 43 nuclear-encoded SSU rDNA and plastid-encoded rbcL gene sequences was generated from the same strains of conjugating green algae (Zygnematophyceae, Streptophyta). Phylogenetic analyses used the genes individually and in combination, either as concatenated sequences or with the log-likelihood summation method. Single-gene analyses, although mostly congruent, revealed some conflicting nodes and showed different patterns of statistical support. Combined analyses confidently resolved the conflicts between the single-gene analyses, enhanced phylogenetic resolution, and were better supported by morphological information. Long-branch taxa were not the same for the two genes analyzed, and, thus, their effect on phylogenetic resolution was minimized in the combined analyses.