Compensatory Base Research Articles

Evaluation of ITS2 molecular morphometrics effectiveness in species delimitation of Ascomycota – A pilot study

Exploring the secondary structure information of nuclear ribosomal internal transcribed spacer 2 (ITS2) has been a promising approach in species delimitation. However, Compensatory base changes (CBC) concept employed in this approach turns futile when CBC is absent. This prompted us to investigate the utility of insertion/deletion (INDELs) and substitutions in fungal delineation at species level. Upon this rationale, 116 strains representing 97 species, belonging to 6 genera (Colletotrichum, Boeremia, Leptosphaeria, Peyronellaea, Plenodomus and Stagonosporopsis) of Ascomycota were retrieved from Q-bank for molecular morphometric analysis. CBC, INDELs and substitutions between the species of their respective genus were recorded. Most species combinations lacked CBC. Among the substitution events, transitions were predominant. INDELs were less frequent than the substitutions. These evolutionary events were mapped upon the helices to discern species specific variation sites. In 68 species unique variation sites were recognised. The remaining 29 species shared absolute similarity with distinctly named species. The variation sites catalogued in them overlapped with other distinct species and resulted in the blurring of species boundaries. Species specific variation sites recognized in this study are the preliminary results and they could be discerned with absolute confidence when larger datasets encompassing all described species of genera were investigated. They could be of potential use in barcoding fungi at species level. This study also concludes that the ITS2 molecular morphometric analysis is an efficient third dimensional study of the fungal species delimitation. This may help to avoid the false positives in species delimitations and to alleviate the challenges in molecular characterization.

Identification and Phylogeny of Cryptic Species of the Daphnia Longispina Complex (Anomopoda, Daphniidae) Using ITS2 Secondary Structure

Cladocerans of the genus Daphnia O.F. Muller (Crustacea: Cladocera) are typical members of different types of water bodies and are used as model organisms in various biological disciplines. Within the genus, a considerable number of closely related and cryptic species are noted, the identification of which by morphological traits often causes difficulties. In the present study, species identification in the Daphnia longispina complex, namely, D. longispina O.F. Muller, D. hyalina Leydig, D. umbra Taylor, Hebert et Colbourne, and D. cf. longispina, was performed using data on the predicted secondary structure of the second internal transcribed spacer (ITS2) of the nuclear rDNA. Phylogenetic analysis carried out using the Profile Neighbor-Joining algorithm and the analysis of compensatory base changes supported the species status of D. hyalina and D. cf. longispina. It was demonstrated that the data on the ITS2 secondary structure can be used to resolve complex taxonomic problems in the study of cladocerans in general and the species complexes in particular.

Internal transcribed spacer 2 (ITS2) molecular morphometric analysis based species delimitation of foliar endophytic fungi from Aglaia elaeagnoidea, Flacourtia inermis and Premna serratifolia.

Molecular morphometrics is an emerging third dimensional aspect of fungal species delimitation. They have been demonstrated to be more informative than conventional barcoding methods. Hence in this study, foliar endophytic fungal (FEF) assemblages in three Magnoliopsida plants were delimited using nuclear ribosomal internal transcribed spacer 2 (ITS2) sequence—secondary structural features based phylogenetic analysis, also known as molecular morphometrics. A total of 392 FEF isolates were obtained from the Aglaia elaeagnoidea, Flacourtia inermis, and Premna serratifolia leaves and grouped into 98 morphotypes. Among these host plants, P. serratifolia showed the maximum percentage of colonization frequency. Representatives of each morphotype was sequenced and subjected to further molecular characterization. The results revealed that morphotypes were belonged to the phylum of Ascomycota, distributed over two classes (Sordariomycetes (68.59%) and Dothideomycetes (31.41%)), 6 orders and 19 genera. Based on compensatory base changes (CBC) analysis and absolute identity of ITS2 structure, 21, 20 and 23 species were recognized from A. elaeagnoidea, F. inermis, and P. serratifolia respectively. Diversity indices were higher in A. elaeagnoidea, despite it accounted for a modest 16.8% of total isolates recorded in this study. The genus Colletotrichum was predominant in A. elaeagnoidea (39%) and P. serratifolia (48%). Similarly, Diaporthe (43%) was dominant in F. inermis. Several host-specific species were also observed. This study concludes that these plants host diverse species of Ascomycota. To the best of our knowledge, this is the first detailed report on FEF diversity from these plants. Also, the inclusion of ITS2 secondary structure information along with the sequence provides a further dimension to resolve the inherent problems in identification of fungal species.

Desmodesmus tropicus (Chlorophyta) in the Danube Delta – reassessing the phylogeny of the series Maximi

ABSTRACT Desmodesmus maximus (West & G.S.West) Hegewald, D. perforatus (Lemmermann) E.Hegewald and D. tropicus (W.B.Crow) E.Hegewald form the series Maximi within the genus Desmodesmus. Desmodesmus tropicus is a rare species although it is widely distributed in tropical regions including South-East Asia, South America and the USA. It is less common in temperate fresh waters, known only from Ukraine. In this study, we report for the first time its presence in the Danube Delta (Puiu Lake), Romania. The morphology and molecular phylogeny of two newly isolated strains were studied in relation to other strains of the genus Desmodesmus, series Maximi. Cell wall structures (rosettes, spines, tubes, honeycomb-shaped mesh with hexagonal units) which are important in the taxonomy of these algae were investigated by light-, scanning- and transmission electron microscopy. The phylogenetic position of the strains in the Algae and Cyanobacteria Culture Collection of the Institute of Biological Research was assessed using 18S rDNA sequences, ITS2 primary and secondary structures and by analysis of Compensatory Base Changes. Morphological traits and measurements of cells, coenobia and spines matched the description of D. tropicus, although some differences from the established morphological key (ridge-like structures resembling fine ribs) were also noticed. Identification as D. tropicus var. longiclathratus (Tell) Jeon & Hegewald was based on the presence of inner spines on four-celled and especially on eight-celled coenobia. The ITS2 sequence and secondary structure phylogeny supported the assignment of both AICB strains to D. tropicus, with no divergence from other D. tropicus sequences to support the identification of var. longiclathratus. Nevertheless, the morphological measurements and the ITS2 primary and secondary structure data indicated two distinct subclusters within D. tropicus, and a new possible taxon within the series Maximi.

Taxonomy and toxicity of Prorocentrum from Perhentian Islands (Malaysia), with a description of a non-toxigenic species Prorocentrum malayense sp. nov. (Dinophyceae)

Thirteen isolates of Prorocentrum species were established from the coral reefs of Perhentian Islands Marine Park, Malaysia and underwent morphological observations and molecular characterization. Six species were found: P. caipirignum, P. concavum, P. cf. emarginatum, P. lima, P. mexicanum and a new morphotype, herein designated as P. malayense sp. nov. Prorocentrum malayense, a species closely related to P. leve, P. cf. foraminosum, P. sp. aff. foraminossum, and P. concavum (Clade A sensu Chomérat et al. 2018), is distinguished from its congeners as having larger thecal pore size and a more deeply excavated V-shaped periflagellar area. Platelet arrangement in the periflagellar area of P. malayense is unique, with the presence of platelet 1a and 1b, platelet 2 being the most anterior platelet, and a broad calabash-shaped platelet 3. The species exhibits consistent genetic sequence divergences for the nuclear-encoded large subunit ribosomal RNA gene (LSU rDNA) and the second internal transcribed spacer (ITS2). The phylogenetic inferences further confirmed that it represents an independent lineage, closely related to species in Clade A sensu Chomérat et al. Pairwise comparison of ITS2 transcripts with its closest relatives revealed the presence of compensatory base changes (CBCs). Toxicity analysis showed detectable levels of okadaic acid in P. lima (1.0–1.6 pg cell˗1) and P. caipirignum (3.1 pg cell˗1); this is the first report of toxigenic P. caipirignum in the Southeast Asian region. Other Prorocentrum species tested, including the new species, however, were below the detection limit.

Fritschiella aquatilis (Chaetophoraceae, Chlorophyta), a new freshwater green algae species from China

A new freshwater algal species of Fritschiella aquatilis was found in a freshwater aquarium in China (Shanxi Province), floating on the surface or sinking at the bottom of the water. This new species is characterized by bright-green, globular thallus with hairy morphology, which composed numerous small single celled filamentous branches. Most characteristics are in agreement with description of Fritschiella. But the new species differs from other members in this genus by the particularly aquatic habitat and macroscopic thalli morphology (the only reported Fritschiella tuberosa Iyengar species is terrestrial). Concatenated sequences from 28S rDNA, 18S rDNA, rbcL gene phylogenetic analysis of F. aquatilis to discuss taxonomic status. All phylogenetic trees have yielded a Fritschiella clade included the sequence, and F. aquatilis formed an independent branch evidently distinguished from other species. Comparing with F. tuberosa, the internal transcribed spacer (ITS) secondary structure of the new species has some compensatory base changes (CBCs) and hemi-CBCs, especially at the Helix1 and Helix 2 end. Thus, the results of morphological and molecular characteristics support F. aquatilis to be recognized as a new species. This new alga brings the total number of recognized Fritschiella species, and the habitats circumscription of the genus Fritschiella expanded to include terrestrial and aquatic.

Application of Morphological and Molecular Systematics for Identification of Green Microalgae of the Genus Chlorococcum and Some Closely Related Taxa

Polyphyletic nature of three genera of green microalgae, Chlorococcum, Deasonia, and Neospongiococcum, was confirmed. Some Chlorococcum species, together with the type species C. infusionum, formed a true clade Chlorococcum within the macroclade Moewusinia. Its members were characterized by extremely long ITS2 regions with unusual secondary structure. Other species were found to belong to the Stephanosphaerinia macroclade, and should be reclassified accordingly. The research of authentic strains resulted in revised and enhanced morphological descriptions of N. gelatinosum, C. diplobionticum, C. isabeliense, and C. sphacosum. Proposals on taxonomic revision are based on morphological similarity, 18S rRNA distances, presence of compensatory base changes in ITS2 conserved regions, differences in the secondary structure of the spacer, and differences of the helix III conserved motif.

Watanabea acidotolerans: A new trebouxiophyte lineage (Chlorophyta) inhabiting low pH environments from Europe to South America

SUMMARYHere we studied a new Watanabea strain CAUP H 8901‐CRYO isolated from an extremely acidic (pH 2.6) pit lake in Czechia. We used morphological, ultrastructural and molecular data to ascertain its taxonomic position and phylogenetic relationships within the genus Watanabea. The morphology of the studied strain matched the formal Watanabea description, including its most characteristic feature, the production of two types of autospores. The new isolate morphologically and ultrastructurally resembled the Watanabea type species, W. reniformis. However, it differed from W. borysthenica by having a pyrenoid not surrounded by starch grains. Molecular phylogenetic analyses of nuclear 18S rDNA and plastid rbcL showed that the strain CAUP H 8901‐CRYO formed an independent lineage within the genus Watanabea. Furthermore, analyses of nuclear ITS2 secondary structure demonstrated an additional side loop coiling from the helix III, the unique feature of CAUP H 8901‐CRYO, not present in the authentic strain of W. reniformis SAG 211‐9b. The ITS2 secondary structures of the two strains also differed by one compensatory base change (CBC) and a number of additional nucleotide substitutions. The new acidotolerant strain and another Watanabea isolate from a naturally acidic lake in Argentina were identical in their plastid rbcL sequence. Consequently, here we proposed a new species, Watanabea acidotolerans, to accommodate a lineage of microalgae with a possible worldwide distribution and tolerance to low pH habitats.

Identification and characterization of novel conserved RNA structures in Drosophila

BackgroundComparative genomics approaches have facilitated the discovery of many novel non-coding and structured RNAs (ncRNAs). The increasing availability of related genomes now makes it possible to systematically search for compensatory base changes – and thus for conserved secondary structures – even in genomic regions that are poorly alignable in the primary sequence. The wealth of available transcriptome data can add valuable insight into expression and possible function for new ncRNA candidates. Earlier work identifying ncRNAs in Drosophila melanogaster made use of sequence-based alignments and employed a sliding window approach, inevitably biasing identification toward RNAs encoded in the more conserved parts of the genome.ResultsTo search for conserved RNA structures (CRSs) that may not be highly conserved in sequence and to assess the expression of CRSs, we conducted a genome-wide structural alignment screen of 27 insect genomes including D. melanogaster and integrated this with an extensive set of tiling array data. The structural alignment screen revealed ∼30,000 novel candidate CRSs at an estimated false discovery rate of less than 10%. With more than one quarter of all individual CRS motifs showing sequence identities below 60%, the predicted CRSs largely complement the findings of sliding window approaches applied previously. While a sixth of the CRSs were ubiquitously expressed, we found that most were expressed in specific developmental stages or cell lines. Notably, most statistically significant enrichment of CRSs were observed in pupae, mainly in exons of untranslated regions, promotors, enhancers, and long ncRNAs. Interestingly, cell lines were found to express a different set of CRSs than were found in vivo. Only a small fraction of intergenic CRSs were co-expressed with the adjacent protein coding genes, which suggests that most intergenic CRSs are independent genetic units.ConclusionsThis study provides a more comprehensive view of the ncRNA transcriptome in fly as well as evidence for differential expression of CRSs during development and in cell lines.

Spongiosarcinopsis terrestris gen. et sp. nov. (Chlorophyta, Chlorophyceae): a new genus of green algae from gray forest soil, Russia

The diversity of soil green microalgae (Chlorophyta) is generally poorly known and likely underestimated because of their morphological poverty and crypticity. The use of molecular tools has revolutionized algal diversity research, including the discovery of numerous new taxa. On the basis of light microscopy, we isolated Chlorochytrium-like strain ACSSI 023 from gray forest soil (Moscow region, Russia), but with spongy rather than network chloroplast. This strain was evaluated by means of 18S rRNA gene and ITS2 sequences analyses and ultrastructural observations. Morphology, comparing the ITS2 secondary structure, compensatory base changes, intron lacking and percent similarity of the 18S rRNA gene of the isolated strain allowed us to propose a new genus and species for it, Spongiosarcinopsis terrestris gen. et sp. nov. The genus forms a separate independent branch within the Stephanosphaerinia clade (Chlorophyta, Chlorophyceae) with type species—Spongiosarcinopsis terrestris ACSSI 023.

Four new species of Bacidia s.s. (Ramalinaceae, Lecanorales) in the Russian Far East

AbstractThe molecular phylogeny of Bacidia s.s. in the Russian Far East was investigated using 62 nucleotide sequences from the ITS nrDNA region, 22 of which were newly obtained. Phylogenetic reconstructions employed Bayesian inference and maximum likelihood searches using MrBayes and RAxML. In addition, ITS2 secondary structures added further support using Compensatory Base Changes. As a result of morphological and phylogenetic studies, four new species of Bacidia are described. Bacidia areolata sp. nov. belongs to the suffusa group. It was collected once in Khabarovskiy Krai, the Russian Far East, on the bark of Acer tegmentosum and is closely related to B. suffusa but differs in having a smooth, cracked to areolate thallus and shorter spores. Bacidia elongata sp. nov. is a member of the fraxinea group and is similar to B. fraxinea but differs in having a wide zone of cells with enlarged lumina along the edge of the exciple. In fact, this zone of enlarged cells, in combination with its overall habit, places it morphologically close to B. suffusa, B. millegrana and B. campalea. Bacidia kurilensis sp. nov. is a basal member of the laurocerasi group and closely related to B. biatorina, B. heterochroa, B. laurocerasi and B. salazarensis. However, the combination of a granular thallus, large black apothecia and a green hue in the upper part of the exciple edge as well as in the epihymenium sets it apart from the species mentioned above. Bacidia sachalinensis sp. nov. resolves as a strongly supported member of the polychroa group and is known from a single locality in Sakhalin, the Russian Far East. Its thallus structure and apothecium colour are variable, which is typical for the polychroa group, but it differs from B. polychroa by having shorter spores with fewer septa and a mainly smooth to areolate thallus.

A consensus secondary structure of ITS2 for the diatom Order Cymatosirales (Mediophyceae, Bacillariophyta) and reappraisal of the order based on DNA, morphology, and reproduction

In 1983, Hasle and colleagues removed cymatosiroid diatoms from the pennates, and erected a new centric diatom family, the Cymatosiraceae, mainly to accommodate for their newly discovered mode of sexual reproduction. The new family consisted of two subfamilies differing in frustule structure. The family was later elevated to the rank of Order Cymatosirales Round and Crawford. We revisited intra-ordinal relationships within Cymatosirales using combined genetic (DNA sequences), morphological (valve and frustule structure), and reproductive (auxospore type) characters. In total, 36 cymatosiroid strains from 19 species representing 13 genera (80% of the total number of extant genera; nine of them represented by their generitypes) were used in this study. Instead of only the commonly used loci (18S rRNA and plastidal genes) to infer diatom phylogeny, we developed a consensus secondary structure model of the Internal Transcribed Spacer 2 (ITS2) for this order and applied it to aid in sequence alignments for ITS2. This improved the alignment and thus the robustness of the phylogenetic framework. The compensatory base changes (CBCs) found in ITS2 secondary structures were mapped onto the multi-gene (18S rRNA + ITS2 + rbcL) phylogenetic tree topology. In all these trees, all species grouped into two morphologically and genetically distinct clades. Each clade was supported by multiple CBCs, as did all the clades representing genera. However, these clades did not correspond to the previously established subfamilies. Consequently, we amend the Order Cymatosirales and family Cymatosiraceae, and propose a new family, the Leyanellaceae. The structure of the auxospore was an additional synapomorphic character for Cymatosirales. Overall, we demonstrate a novel approach to study diatom phylogeny across a broader taxonomic range using ITS2 secondary structural information. Our results suggest that this approach might be useful in establishing higher taxonomic relationships in other groups of diatoms.

Chlamydomonas schloesseri sp. nov. (Chlamydophyceae, Chlorophyta) revealed by morphology, autolysin cross experiments, and multiple gene analyses

Chlamydomonas in the traditional sense is one of the largest green algal genera, comprising more than 500 described species. However, since the designation of the model organism C. reinhardtii as conserved type of this genus in 2007, only two species remained in Chlamydomonas. Investigations of three new strains isolated from soil samples, which were collected near Lake Nakuru (Kenya), demonstrated that the isolates represent a new species of Chlamydomonas. Phylogenetic analyses of nuclear SSU and ITS rDNA and plastid-coding rbcL sequences have clearly revealed that this species is closely related to C. reinhardtii and C. incerta. These results were confirmed by cross experiments of sporangium wall autolysins (VLE). All species belonged to the VLE group 1 sensu Schlösser. The comparison of the ITS-1 and ITS-2 secondary structures showed several compensatory base changes among the three species. In addition, the rbcL amino acid composition was also species-specific. The genus Chlamydomonas was phylogenetically closely related to the colonial families Goniaceae, Tetrabaenaceae and Volvocaceae. Chlamydomonas debaryana (VLE group 2) formed a separate clade among these colonial families of the Volvocales, a species of which autolysin dissolved the sporangium walls of the members of VLE group 1, suggesting its close relationship to Chlamydomonas. As consequence of our results, we propose Chlamydomonas schloesseri sp. nov. for the new Kenyan isolates. We also propose a new combination of C. debaryana to the newly erected genus Edaphochlamys.

Coronacoccus hengyangensis gen. et sp. nov., a new member of Chlorellaceae (Trebouxiophyceae, Chlorophyta) with radiococcacean morphology

:The Chlorellaceae is a family in the order Chlorellales that includes mostly coccoid algae of minute sizes, and a portion of this family was separated into two sister groups (the Chlorella clade and the Parachlorella clade) according to molecular phylogenetic analyses. To date, the Parachlorella clade comprises 11 genera that exhibit a range of morphological variation. In this study, we discovered from China a strain marked as FACHB-2137; we use a polyphasic approach, including phylogenetic analyses of SSU+ITS rDNA, an ITS2/CBC (compensatory base changes) approach, light microscopy and transmission electron microscopy, to characterise the strain. Molecular analyses revealed that the strain was nested within the Parachlorella clade (Chlorellaceae, Trebouxiophyceae), forming an independent lineage separated from other genera. The new strain's morphology has the characteristic autospores that release by rupturing or splitting of the cell wall into several (two to four) portions; this strain differs from other members of the Parachlorella clade in that the mother cell wall remains rather distinct, and the daughter cells remain as cup-shaped in the colonial mucilage for an extended period. Morphologically, this new strain closely resembles members of the Radiococcaceae. This indicates that inferring family-level relationships based exclusively on morphology can be problematic. We describe this alga as a new genus and species, Coronacoccus hengyangensis gen. et sp. nov. This morphotype is described for the first time in the Parachlorella clade.

Dropping the base: Characterizing the hypercarbia recovery strategies following extreme hypercapnia in the highly CO2 tolerant hagfish (Eptatretus stoutii)

As scavengers that feed on decaying carrion (e.g. fish, large marine mammals), hagfish (Eptatretus stoutii) can be subjected to noxious environmental stresses such as high ammonia, anoxia and hypercapnia. While the physiological impacts and the hagfishes' tolerance of these stressors have been well characterized, little data exists on the mechanisms and strategies for recovery from these stressors that would be mandatory in the natural environment. Indeed, hagfish are capable of tolerating extreme hypercapnia (>30 Torr; 72h) by building up plasma [HCO3−] over 24 – 48 h of exposure, attaining the highest plasma [HCO3−] levels ever observed in any organism (~70 mmol HCO3− L−1). The goal of this study was to characterize the hypercarbia recovery strategies of the highly CO2 tolerant following hypercapnia exposure. In a series of experiments, hagfish were exposed to hypercapnia (4% CO2) for 48 h to induce hypercarbia before being allowed to recover in normocapnic seawater. During this recovery period, measurements of blood acid/base status, plasma [Cl−] and net H+/HCO3− flux were made to elucidate the recovery strategies of the hagfish. Upon reintroduction into nonromantic conditions, hagfish rapidly (<8h) offloaded the compensatory base load (65.8 ± 2.1 mmol HCO3− L−1) while sustaining an incredible blood alkalosis of ~0.8 pH units compared to post‐exposure conditions, to a blood pH 8.67 ± 0.03 within 4h of recovery. While increases in both whole‐animal HCO3− excretion and glomerular filtration were observed throughout recovery (2–8 h), neither can fully account for the observed rates of whole‐animal HCO3− loss, which peaked at ~3.5 mmol kg−1 h−1. Inhibition of carbonic anhydrase using acetazolamide revealed that restoration of plasma [HCO3−] from hypercapnia‐induced hypercarbia is likely facilitated in a dualistic manner, initially relying on both carbonic anhydrase mediated CO2 offloading and Cl−/HCO3− exchange processes, both of which are likely either upregulated or further activated as recovery progresses.Support or Funding InformationNSERC (GGG: 203736)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A new freshwater alga, Ulvella shanxiensis (Chlorophyta) described from China

A new freshwater alga species of Ulvella shanxiensis, is found in the circulating neutral water spring in China (Shanxi Province), growing epilithic and epiphytic on other substrates. This new species is characterized by olive-green, disc-shaped hollow thallus with irregularly vesicular morphology, which composed of 2–3 layers cells. Most characteristics were in agreement with Ulvella, but distinguished from the other freshwater members by thalli dimensions and cell diameter. In addition to describing the morphological structures of U. shanxiensis in detail, phylogenetic analysis based on sequences of the rbcL and 18S rDNA gene placed the algae in single clade with two samples, and demonstrated the separation between U. shanxiensis and other Ulvella species with a considerable sequence distance. Comparing with U. bullata, the internal transcribed spacer secondary structure of the new species has some compensatory base changes in 1 Helix and hemi-CBCs in 2–3 Helix, differed in several ways from that of other Ulvella algae. Consequently, these results of morphological observation and phylogenetic analysis suggest this alga as a new species, and bring a new record in the total number of recognized freshwater Ulvella species in China.

ITS2 sequence-structure phylogeny reveals diverse endophytic Pseudocercospora fungi on poplars.

For matching the new fungal nomenclature to abolish pleomorphic names for a fungus, a genus Pseudocercospora s. str. was suggested to host holomorphic Pseudocercosproa fungi. But the Pseudocercosproa fungi need extra phylogenetic loci to clarify their taxonomy and diversity for their existing and coming species. Internal transcribed spacer 2 (ITS2) secondary structures have been promising in charactering species phylogeny in plants, animals and fungi. In present study, a conserved model of ITS2 secondary structures was confirmed on fungi in Pseudocercospora s. str. genus using RNAshape program. The model has a typical eukaryotic four-helix ITS2 secondary structure. But a single U base occurred in conserved motif of U-U mismatch in Helix 2, and a UG emerged in UGGU motif in Helix 3 to Pseudocercospora fungi. The phylogeny analyses based on the ITS2 sequence-secondary structures with compensatory base change characterizations are able to delimit more species for Pseudocercospora s. str. than phylogenic inferences of traditional multi-loci alignments do. The model was employed to explore the diversity of endophytic Pseudocercospora fungi in poplar trees. The analysis results also showed that endophytic Pseudocercospora fungi were diverse in species and evolved a specific lineage in poplar trees. This work suggested that ITS2 sequence-structures could become as additionally significant loci for species phylogenetic and taxonomic studies on Pseudocerospora fungi, and that Pseudocercospora endophytes could be important roles to Pseudocercospora fungi's evolution and function in ecology.

Chloromonas arctica sp. nov., a psychrotolerant alga from snow in the High Arctic (Chlamydomonadales, Chlorophyta).

With the advent of molecular phylogenetic methods, it has become possible to assess the bioversity of snow algae more accurately. In this study, we focused on a morphological, ultrastructural and taxonomic description of a new Chloromonas-like alga isolated from snow in the High Arctic (Svalbard). Light and transmission electron microscopy revealed broad ellipsoidal or ellipsoidal-cylindrical, occasionally spherical cells with a chloroplast without a pyrenoid, an inconspicuous eyespot and a papilla. The size difference and the aforementioned morphological traits clearly distinguished the alga from its closest counterparts within the genus Chloromonas. Moreover, we were able to cultivate the alga at both 5 and 20 °C, revealing the psychrotolerant nature of the strain. Phylogenetic analyses of the plastid rbcL and nuclear 18S rRNA gene showed that the alga is nested within a clade containing a number of psychrotolerant strains within the Chloromonadinia phylogroup (Chlorophyceae). In the rbcL phylogeny, the alga formed an independent lineage, sister to the freshwater species Chloromonas paraserbinowii. Comparisons of secondary structure models of a highly variable ITS2 rDNA marker showed support for a distinct species identity for the new strain. The ITS2 secondary structure of the new isolate differed from the closest matches 'Chlamydomonas' gerloffii and Choloromonas reticulata by three and five compensatory base changes, respectively. Considering the morphological and molecular differences from its closest relatives, a new psychrotolerant species from the Arctic, Choromonas arctica sp. nov., is proposed.

Biecheleria tirezensis sp. nov. (Dinophyceae, Suessiales), a new halotolerant dinoflagellate species isolated from the athalassohaline Tirez natural pond in Spain

ABSTRACTA new euryhaline and eurythermal dinoflagellate species, Biecheleria tirezensis sp. nov., is described based on samples taken from an extreme environment, the athalassohaline and particularly sulphate-rich Tirez natural pond (Spain). This species is able to survive in salinities from almost fresh water up to 56 and over a 5–25°C temperature range. Thus, the ecological characteristics of this isolate differentiate it from other species of the same genus. Its morphology, as examined by light, scanning and transmission electron microscopy, shows that the cells are almost spherical, with several series of amphiesmal vesicles. It also has a single elongate amphiesmal vesicle (EAV) observed by SEM and the eyespot has a type E structure, typical of Biecheleria. Spherical and smooth cysts were observed in old cultures. The pigment composition is typical of a peridinin-containing dinoflagellate. Phylogeny inferred from nuclear rDNA SSU, ITS and LSU sequences showed the isolate belongs to the genus Biecheleria, closest to B. cincta and B. brevisulcata. Modelling and analysis of the secondary structure of its ITS2 region, and that of other species of the same genus and some representatives of the most closely related genera, indicated that the isolate represents a new species clearly separated from but related to B. cincta. The criterion of the presence of Compensatory Base Changes (CBCs) in the secondary structure of the ITS2 region as an indicator of species differentiation confirmed this, supporting the establishment of the Tirez pond isolate as a new species of Biecheleria.

New finding of green algae with potential for algal biotechnology, Chlorococcum oleofaciens and its molecular investigation

The practice of soil algology shows that algae from the order Chlamydomonadales are among the most poorly studied and difficult to identify due to the high heterogeneity of their morphology and ultrastructure. Only the involvement of molecular genetic methods usually makes it possible to determine their taxonomic status with high accuracy. At the same time, in the algae flora of Ukraine there are more than 250 species from the order Chlamydomonadales, the status of which in most cases is established exclusively on the basis of light microscopy. This work is devoted to the study of the biotechnologically promising green alga Chlorococcum oleofaciens, taking into account the modern understanding of its taxonomic status. Two new strains of this species, separated from samples of forest litter and oak forest soil (the Samara Forest, Dnipropetrovsk region), are described. The strains were studied at the morphological level by using light microscopy methods, as well as using molecular genetic methods based on the studies of the nucleotide genes sequences of the 18S ribosomal DNA (rDNA) and chloroplast rbcL genes, the topology of secondary structures of internal transcribed spacer 2 (ITS2). The obtained results helped to confirm the presence of C. oleofaciens in the algae flora of Ukraine. Also, the authors of the article discuss the differences in the secondary structure of ITS2 in different strains of C. oleofaciens associated with the presence of compensatory base change (CBC), hemi-CBC in helices I and II, as well as deletions in helix IV and providing a basis for the hypothesis of the existence of cryptic species within C. oleofaciens. The obtained data can be used at the stage of preliminary selection of biochemical research objects.