Molecular characterization of Cylindrospermopsis raciborskii strains isolated from Portuguese freshwaters

Cylindrospermopsis raciborskii is a toxic bloom forming cyanobacteria that is a common component of the phytoplankton assemblage in temperate freshwaters, as well as in temperate climates. This species is of major concern in public health, due to its known ability to produce toxins, including cylindrospermopsin and paralytic shellfish poisoning toxin (PSP).In this study, M13 PCR fingerprinting, ERIC PCR fingerprinting and amplification of the internal transcribed spacer (ITS) region were used to characterize nine cultured strains of C. raciborskii, sourced from several freshwater lakes and rivers in Portugal, and two other Australian. Strains belonging to other taxa including Microcystis aeruginosa, Aphanizomenon spp., Planktothrix agardhii and Oscillatoria neglecta were also analysed to evaluate the taxonomical potential of the fingerprinting methods.Data obtained from genomic fingerprinting were used to perform hierarchical cluster analysis and demonstrated ability to differentiate strains at intra-specific level. However, the high level of variability prevents their use as an identification tool. ITS amplification displayed intra-specific polymorphism both in number and length of the obtained amplicons, but revealed itself as a good method for strain clustering. The unsuccessful amplification of peptide synthetase (PS) and polyketide synthase (PKS) genes pointed to the inability of Portuguese C. raciborskii strains to produce cylindrospermopsin. HPLC analysis further confirmed this lack of toxicity, since negative results were obtained for cylindrospermopsin and PSP toxins.     

Molecular phylogeny and phenotypic variability of clinical and environmental strains of Aspergillus flavus

Aspergillus flavus is the second most common cause of aspergillosis infection in immunocompromised patients and is responsible for the production of aflatoxins. Little is known about the population structure of A. flavus, although recent molecular and phenotypic data seem to demonstrate that different genetic lineages exist within this species. The aim of this study was to carry out a morphological, physiological, and molecular analysis of a set of clinical and environmental isolates to determine whether this variability is due to species divergence or intraspecific diversity, and to assess whether the clinical isolates form a separate group. The amdS and omtA genes were more phylogenetically informative than the other tested genes and their combined analysis inferred three main clades, with no clear distinction between clinical and environmental isolates. No important morphological and physiological differences were found between the members of the different clades, with the exception of the assimilation of d-glucosamine, which differentiates the members of the clade II from the others.     

On the systematics of some marine haploporids (Trematoda) with the description of a new species of Megasolena Linton, 1910

Megasolena mikra sp. nov. is described from the queen angelfish, Holacanthus ciliaris (Linnaeus), off Florida, USA. The new species can be differentiated from all other species of Megasolena Linton, 1910 except Megasolena littoralis Muñoz, George-Nascimento, and Bray, 2017 in possessing testes that are smaller in diameter than the ovary. The new species can be differentiated from M. littoralis in lacking tegumental spines and possessing oral sucker papillae. Molecular data are provided for two species each of Cadenatella Dollfus, 1946, Hapladena Linton, 1910, and Megasolena Linton, 1910. Bayesian inference analysis of concatenated internal transcribed spacer region-2 (ITS2) and partial 28S rDNA sequences of 50 haploporoids revealed 1) a monophyletic Atractotrematidae Yamaguti, 1939 sister to the rest of the haploporoids tested; 2) a paraphyletic Megasoleninae Manter, 1935 – if Hapladena is included; and 3) a monophyletic Cadenatellinae Gibson and Bray, 1982 sister to the ‘mugilid’ haploporids. The ‘mugilid’ haploporids formed a monophyletic clade consisting of the subfamilies Chalcinotrematinae Overstreet and Curran, 2005, Forticulcitinae Blasco-Costa, Balbuena, Kostadinova, and Olson, 2009, Haploporinae Nicoll, 1914, and Waretrematinae Srivastava, 1937. Based on our analysis we restrict the Megasoleninae to include Megasolena, Vitellibaculum Montgomery, 1957, and Metamegasolena Yamaguti, 1970, all of which have species with two testes. To accommodate the former megasolenine taxa with a single testis, we erect the Hapladeninae subf. nov. for species in Hapladena and tentatively, Myodera Montgomery, 1957. Our results further support that haploporoids had a common marine ancestor with two testes, and that members of the Haploporoidea Nicoll, 1914 underwent diversification following a shift from a primarily marine life history with eupercarian hosts to a more euryhaline one with diadromous hosts (namely mullet).     

Molecular phylogeny of Collembola inferred from ribosomal RNA genes

The classification of taxa within Collembola (Springtails, Hexapoda) has been controversial. In this study, we combined complete 18S rRNA gene with partial 28S rRNA gene (D7-D10) sequences to investigate the phylogeny of Collembola. About 2500 aligned sites of thirty species representing 29 genera from14 families of Collembola were analyzed, including one species of Neelipleona from which no sequence has been reported previously.The phylogenetic trees were obtained by different methods (maximum parsimony, maximum likelihood, and Bayesian analysis). Our results supported the monophyly of two of the four taxonomic groups of Collembola summarized by Deharveng [Deharveng, L., 2004. Recent advances in Collembola systematics. Pedobiologia 48, 415–433.], namely of Poduromorpha and of Symphypleona. Within Poduromorpha, Neanuridae was monophyletic with high support, but Hypogastruridae was not. Entomobryomorpha was paraphyletic, as the Tomoceroidea (Tomoceridae and Oncopoduridae) was found to be apart from the other entomobryomorphs. In the latter Isotomoidea and Entomobryoidea joined into a group with moderate support. Within Symphypleona, the phylogenetic relationship [(Sminthuridae + Bourletiellidae) + Sminthurididae] was consistent with traditional morphological studies. Neelipleona grouped with Symphypleona in all trees, with moderate support in the ML and Bayesian analyses.     

Application of targeted metagenomics to explore abundance and diversity of CO2-fixing bacterial community using cbbL gene from the rhizosphere of Arachis hypogaea

Sequestration of CO₂ by autotrophic bacteria is a key process of biogeochemical carbon cycling in soil ecosystem. Rhizosphere is a rich niche of microbial activity and diversity, influenced by change in atmospheric CO₂. Structural changes in rhizosphere composition influence microbial communities and the nutrient cycling. In the present study, the bacterial diversity and population dynamics were established using cbbL and 16S rRNA gene targeted metagenomics approach from the rhizosphere of Arachis hypogaea. A total of 108 cbbL clones were obtained from the rhizospheric soil which revealed predominance of cbbL sequences affiliated to Rhizobium leguminosarum, Bradyrhizobium sp., Sinorhizobium meliloti, Ochrobactrum anthropi and a variety of uncultured cbbL harboring bacteria. The 16S rRNA gene clone library exhibited the dominance of Firmicutes (34.4%), Proteobacteria (18.3%), Actinobacteria (17.2%) and Bacteroidetes (16.1%). About 43% nucleotide sequences of 16S rRNA gene clone library were novel genera which showed < 95% homology with published sequences. Gene copy number of cbbL and 16S rRNA genes, determined by quantitative real‐time PCR (qRT PCR), was 9.38 ± 0.75 × 10⁷ and 5.43 ± 0.79 × 10⁸ (per g dry soil), respectively. The results exhibited bacterial community structure with high bacterial diversity and abundance of CO₂‐fixing bacteria, which can be explored further for their role in carbon cycling, sustainable agriculture and environment management.     

Genomic and phenotypic heterogeneity of Acidithiobacillus spp. strains isolated from diverse habitats in China

The genetic variability among 32 Chinese Acidithiobacillus spp. environmental isolates and four reference strains representing three recognized species of the genus Acidithiobacillus was characterized by using a combination of molecular methods, namely restriction fragment length polymorphisms of PCR-amplified 16S rRNA genes and 16S-23S rRNA gene intergenic spacers, repetitive element PCR, arbitrarily primed PCR and 16S rRNA gene sequence analyses. 16S rRNA gene sequences revealed that all Acidithiobacillus spp. strains could be assigned to seven groups, three of which encompassed the Acidithiobacillus ferrooxidans strains from various parts of the world. A comparative analysis of the phylogenetic Group 1 and 2 was undertaken. Restriction fragment length polymorphism results allowed us to separate the 35 Acidithiobacillus strains into 15 different genotypes. An integrated phenotypic and genotypic analysis indicated that the distribution of A. ferrooxidans strains among the physiological groups were in agreement with their distribution among the genomic groups, and that no clear correlation was found between the genetic polymorphism of the Acidithiobacillus spp. strains and either the geographic location or type of habitats from which the strains were isolated. In addition, five unidentified sulfur-oxidizing isolates may represent one or two novel species of the genus Acidithiobacillus. The results showed that the Chinese Acidithiobacillus spp. isolates exhibited a high degree of genomic and phenotypic heterogeneity.     

Evaluation of four molecular typing methodologies as tools for determining taxonomy relations and for identifying species among Yersinia isolates

In the last few decades, molecular typing has become an important tool in taxonomic, phylogenetic and identification studies of numerous groups of bacteria, including the yersiniae. In this study, Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR), Pulsed-Field Gel Electrophoresis (PFGE), 16S rRNA gene sequencing and Multilocus Sequence Analysis (MLSA) were performed to determine the ability of these techniques to be used in taxonomy and identification of Yersinia strains. A total of 60 Yersinia strains were genotyped by ERIC-PCR and PFGE. Moreover, an in silico analysis was carried out for 16S rRNA gene sequencing and MLSA, using 68 and 49 Yersinia strains, respectively. A phylogenetic tree constructed from the ERIC-PCR, 16S rRNA gene sequencing and MLSA data grouped most of the Yersinia species into distinct species-specific clusters. In the PFGE assay these clusters were not observed. On this basis, ERIC-PCR, 16S rRNA gene sequencing and MLSA seem to be valuable techniques for use in taxonomic and identification studies of the genus Yersinia, whereas PFGE does not. Furthermore, ERIC-PCR has the advantage of being a cheaper, easier and faster assay than 16S rRNA gene sequencing or MLSA, and for these reasons can be considerate an alternative tool in taxonomic studies of yersiniae.     

Phylogenic homogeneity of Coxiella burnetii strains as determinated by 16S ribosomal RNA sequencing

Abstract DNA coding for the 16S rRNA of six strains of the obligate intracellular bacterium Coxiella burnetti was directly amplified from lysed host cells using the polymerase chain reaction. The amplification product was sequenced using a linear-PCR procedure and compared with other published 16S rRNA sequences. The results of this analysis confirm the position of C. burnetii in the gamma subgroup of the proteobacteria. The data show that all of the C. burnetii strains are highly related (> 99%) on the basis of 16S rRNA sequences although they had different geographic origins and phenotypic characteristics. The data support a phylogenetic homogeneity of the genus Coxiella with only one species which is C. burnetii .     

Coral reef invertebrate microbiomes correlate with the presence of photosymbionts

Coral reefs provide habitat for an array of marine invertebrates that host symbiotic microbiomes. Photosynthetic symbionts including Symbiodinium dinoflagellates and diatoms potentially influence the diversity of their host-associated microbiomes by releasing carbon-containing photosynthates and other organic compounds that fuel microbial metabolism. Here we used 16S ribosomal RNA (rRNA) gene amplicon pyrosequencing to characterise the microbiomes of 11 common Great Barrier Reef marine invertebrate species that host photosynthetic symbionts and five taxa in which they are absent. The presence of photosynthetic symbionts influenced the composition but not the species richness, evenness and phylogenetic diversity of invertebrate-associated microbiomes. Invertebrates without photosynthetic symbionts were dominated by Alphaproteobacteria, whereas those hosting photosynthetic symbionts were dominated by Gammaproteobacteria. Interestingly, many microbial species from photosymbiont-bearing invertebrates, including Oceanospirillales spp., Alteromonas spp., Pseudomonas spp., Halomonas spp., are implicated in the metabolism of dimethylsulfoniopropionate (DMSP). DMSP is produced in high concentrations by photosynthetic dinoflagellates and is involved in climate regulation by facilitating cloud formation. Microbiomes correlated with host taxa and replicate individuals from most sampled species grouped in distance-based redundancy analysis of retrieved 16S rRNA gene sequences. This study highlights the complex nature of invertebrate holobionts and confirms the importance of photosynthetic symbionts in structuring marine invertebrate bacterial communities.     

Presence of a novel 16S-23S rRNA gene intergenic spacer insert in Bradyrhizobium canariense strains

Seven slow-growing bacterial strains isolated from root nodules of yellow serradella (Ornithopus compressus) that originated from Asinara Island on North Western Sardinia in Italy were characterized by partial 16S rRNA gene and intergenic spacer (ITS) sequencing as well as amplified fragment length polymorphism (AFLP) genomic fingerprinting. The results indicated that the O. compressus isolates belong to the Bradyrhizobium canariense species. The analysis of ITS sequences divided the branch of B. canariense strains into two statistically separated groups (ITS clusters I and II). All the strains in ITS cluster I showed the presence of unique oligonucleotide insert TTAGAGACTTAGGTTTCTK. This insert was neither found in other described species of the family Rhizobiaceae nor in any other bacterial families and can be used as a natural and high selective genetic marker for ITS cluster I of B. canariense strains. ITS grouping of O. compressus isolates was supported by the unweighted pair group method with arithmetic averages cluster analysis of their AFLP patterns, suggesting that the strains of ITS cluster II were genetically closer to each other than to isolates from the ITS cluster I. A taxonomic importance is supposed of the revealed 19 bp ITS insert for an intraspecific division within high heterogeneous B. canariense species.     

Molecular phylogeny and biogeography of the narrow endemic Coelonema and affinitive Draba (Brassicaceae) based on two DNA regions

To clarify the relationship between two genera, Draba and the narrow genus Coelonema, endemic to the QilianMountains of the northeastern Qinghai-Tibet Plateau, phylogenetic analyses were conducted using nuclear ribosomal DNA ITS, and the chloroplast DNA trnL, from Coelonema draboides and 30 species of Draba representing eight sections, including 25 species of Chinese Draba, seven of which were endemic to the study region. The results unambiguously support several previously published proposals to unite Coelonema with Draba and accommodate C. draboides in the latter genus on the basis of morphological re-examination. Our molecular data presented here also provide evidence that these two genera should be combined as a monophyletic group with high support. In addition, it is estimated that Draba may have originated about 1.36–2.71 Mya, with C. draboides diverging from Draba about 0.15–0.31 Mya, based on the molecular calibration of ITS datasets. The assumed speciation and rapid expansion of these two genera is likely to have occurred in the eastern edge of the QilianMountains area according to molecular phylogeny and estimated divergence times, which correspond well with the known geological and paleobotanical histories of the Qinghai-Tibet Plateau.     

Genetic evidence for three discrete taxa of Melampsora (Pucciniales) affecting willows (Salix spp.) in New York State

Rust fungi in the genus Melampsora (Pucciniales) are the most important pathogens of shrub willows (Salix spp.) cultivated for biomass in New York State and temperate regions worldwide. The taxonomy and species identification of these fungi historically have been problematic as they are morphologically indistinguishable on willow and often have complex life histories. Melampsora of Salix in North America, therefore, have been circumscribed to the collective species Melampsora epitea Thüm. and further delineated to formae speciales by aecial host. Ribosomal DNA (rDNA) data was obtained from 75 collections/isolates of Melampsora in NY State affecting either native and cultivated Salix spp. or suspected alternate hosts. Maximum likelihood (ML), maximum parsimony (MP), and Bayesian (BI) analyses were conducted on three data partitions (individual and concatenated): complete internal transcribed spacer (ITS) and partial large subunit (LSU) rDNA sequences for all collections. Analyses of the ITS and concatenated ITS-LSU sequences revealed that Melampsora on native and cultivated willows in NY State consisted of three phylogenetically delineable taxa (phylotaxa); monophyly for each phylotaxon was strongly supported by ML, MP, and BI credibility values. Phylotaxa were also delimited phylogenetically by aecial host: Alpine currant (Ribes alpinum), eastern larch (Larix laricina), or balsam fir (Abies balsamea).     

Genotypic and phenotypic variation among Lysobacter capsici strains isolated from Rhizoctonia suppressive soils

Four Gram-negative bacterial strains, recovered from clay soils cultivated with different crops in the Netherland, were subjected to a polyphasic taxonomic study in order to clarify their taxonomic status. Comparative analysis of the 16S rRNA gene sequences revealed that they belong to the genus Lysobacter and to be highly related to the type strains of L. antibioticus DSM 2044^T, L. gummosus DSM 6980^T, and L. capsici DSM 19286^T, displaying 99.1–99.3%, 99.2–99.6% and 99.4–100% sequence similarities, respectively, to these species. The results of DNA–DNA hybridization studies unambigiously indicated that the four strains belonged to the species L. capsici. Nevertheless, DNA fingerprinting and phenotypic characterization indicated that there was a considerable diversification and niche differentiation among the strains belonging to L. capsici. The newly identified L. capsici strains strongly inhibit Rhizoctonia solani AG2 and originate from Rhizoctonia-suppressive soils where also populations of L. antibioticus and L. gummosus were present. This is the first report of the presence of combined populations of closely related Lysobacter spp. within agricultural soils.     

Raphidiopsis mediterranea Skuja represents non-heterocytous life-cycle stages of Cylindrospermopsis raciborskii (Woloszynska) Seenayya et Subba Raju in Lake Kastoria (Greece), its type locality: Evidence by morphological and phylogenetic analysis

The taxonomical relationship of Cylindrospermopsis raciborskii and Raphidiopsis mediterranea was studied by morphological and 16S rRNA gene diversity analyses of natural populations from Lake Kastoria, Greece. Samples were obtained during a bloom (23,830 trichomes mL⁻¹) in August 2003. A high diversity of apical cell, trichome, heterocyte and akinete morphology, trichome fragmentation and reproduction was observed. Trichomes were grouped into three dominant morphotypes: the typical and the non-heterocytous morphotype of C. raciborskii and the typical morphotype of R. mediterranea. A morphometric comparison of the dominant morphotypes showed significant differences in mean values of cell and trichome sizes despite the high overlap in the range of the respective size values. Additionally, two new morphotypes representing developmental stages of the species are described while a new mode of reproduction involving a structurally distinct reproductive cell is described for the first time in planktic Nostocales. A putative life-cycle, common for C. raciborskii and R. mediterranea is proposed revealing that trichome reproduction of R. mediterranea gives rise both to R. mediterranea and C. raciborskii non-heterocytous morphotypes. The phylogenetic analysis of partial 16S rRNA gene (ca. 920 bp) of the co-existing Cylindrospermopsis and Raphidiopsis morphotypes revealed only one phylotype which showed 99.54% similarity to R. mediterranea HB2 (China) and 99.19% similarity to C. raciborskii form 1 (Australia). We propose that all morphotypes comprised stages of the life cycle of C. raciborkii whereas R. mediterranea from Lake Kastoria (its type locality) represents non-heterocytous stages of Cylindrospermopsis complex life cycle.