Ultrastructural characterisation of cells specialised for nitrogen fixation in a non-heterocystous cyanobacterium,Trichodesmium spp.

Summary Trichodesmium is the first described example of a filamentous cyanobacterium without heterocysts that contains cells specialised for nitrogen fixation. The ultrastructure of cells with and without nitrogenase were compared using primarilyTrichodesmium tenue Wille, but alsoT. thiebautii Gomont andT. erythraeum Ehrenberg et Gomont. Immunohistochemistry demonstrated that the cytoplasm of certain cells was densely labelled with antibodies against Fe-protein (dinitrogenase reductase). Comparative TEM-image analysis revealed that these cells were also distinguished by a denser thylakoid network, dividing the vacuole-like space into smaller units. The nitrogenase-containing cells also exhibited less extensive gas vacuoles as well as fewer and smaller cyanophycin granules compared to cells which lacked nitrogenase. Carboxysomes were present in both cell types in equal proportion. Longitudinal sections showed that cells with nitrogenase were arranged adjacent to each other, and that groups of cells with and without nitrogenase may coexist in the same trichome. The correlation between modifications in ultrastructure and the presence of nitrogenase suggests a new type of cyanobacterial cell specialisation related to nitrogen fixation. The results obtained also question the systematic affiliation of the genusTrichodesmium.     

Characterization of Trichodesmium spp. by Genetic Techniques

The genetic diversity of Trichodesmium spp. from natural populations (off Bermuda in the Sargasso Sea and off North Australia in the Arafura and Coral Seas) and of culture isolates from two regions (Sargasso Sea and Indian Ocean) was investigated. Three independent techniques were used, including a DNA fingerprinting method based on a highly iterated palindrome (HIP1), denaturing gradient gel electrophoresis of a hetR fragment, and sequencing of the internal transcribed spacer (ITS) of the 16S-23S rDNA region. Low genetic diversity was observed in natural populations of Trichodesmium spp. from the two hemispheres. Culture isolates of Trichodesmium thiebautii, Trichodesmium hildebrandtii, Trichodesmium tenue, and Katagnymene spiralis displayed remarkable similarity when these techniques were used, suggesting that K. spiralis is very closely related to the genus Trichodesmium. The largest genetic variation was found between Trichodesmium erythraeum and all other species of Trichodesmium, including a species of Katagnymene. Our data obtained with all three techniques suggest that there are two major clades of Trichodesmium spp. The HIP1 fingerprinting and ITS sequence analyses allowed the closely related species to be distinguished. This is the first report of the presence of HIP1 in marine cyanobacteria.     

PHYLOGENY AND GENETIC VARIANCE IN TERRESTRIAL MICROCOLEUS (CYANOPHYCEAE) SPECIES BASED ON SEQUENCE ANALYSIS OF THE 16S rRNA GENE AND ASSOCIATED 16S–23S ITS REGION1

Thirty‐one strains of Microcoleus were isolated from desert soils in the United States. Although all these taxa fit the broad definition of Microcoleus vaginatus (Vaucher) Gomont in common usage by soil algal researchers, sequence data for the 16S rRNA gene and 16S–23S internal transcribed spacer (ITS) region indicated that more than one species was represented. Combined sequence and morphological data revealed the presence of two morphologically similar taxa, M. vaginatus and Microcoleus steenstrupii Boye‐Petersen. The rRNA operons of these taxa were sufficiently dissimilar that we suspect the two taxa belong in separate genera. The M. vaginatus clade was most similar to published sequences from Trichodesmium and Arthrospira. When 16S sequences from the isolates we identified as M. steenstrupii were compared with published sequences, our strains grouped with M. chthonoplastes (Mertens) Zanardini ex Gomont and may have closest relatives among several genera in the Phormidiaceae. Organization within the 16S–23S ITS regions was variable between the two taxa. Microcoleus vaginatus had either two tRNA genes (tRNA^Ile and tRNA^Ala) or a fragment of the tRNA^Ile gene in its ITS regions, whereas M. steenstrupii had rRNA operons with either the tRNA^Ile gene or no tRNA genes in its ITS regions. Microcoleus vaginatus showed no subspecific variation within the combined morphological and molecular characterizations, with 16S similarities ranging from 97.1% to 99.9%. Microcoleus steenstrupii showed considerable genetic variability, with 16S similarities ranging from 91.5% to 99.4%. In phylogenetic analyses, we found that this variability was not congruent with geography, and we suspect that our M. steenstrupii strains represent several cryptic species.     

ULTRASTRUCTURE AND IMMUNOLOCALIZATION OF PHYCOBILIPROTEINS AND RIBULOSE 1,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE IN THE MARINE CYANOBACTERIUM TRICHODESMIUM THIEBAUTII1

Trichodesmium thiebautii Gomont, a marine planktonic diazotrophic cyanobacterium, has an unusual subcellular arrangement. To identify subcellular structures related to photosynthesis, antibodies against phycoerythrin, phycocyanin, and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) were used together with an immuno-gold labeling technique and electron microscopy. Thylakoid membranes, identified by transmission electron microscopy and phycobiliprotein labeling, were arranged as a loose network throughout all cells. Rubisco showed a particularly intense localization in medium electron-dense polyhedral bodies, therefore identified as carboxysomes. The average density of the carboxysomal Rubisco label was about five times higher than that in the cytoplasm. The carboxysomes (4–11 per cell section) were scattered throughout the cytoplasm. These data, together with those obtained from double immunolabeling experiments using nitrogenase (Fe-protein) and Rubisco antibodies, revealed that Trichodesmium contains both N₂- and CO₂-fixing proteins within the same cell. This is in contrast to the previous concept of a spatial segregation of the two processes in Trichodesmium and demonstrate that nitrogenase-containing cells are not comparable to heterocysts in this context.     

A molecular assessment of the iron stress response in the two phylogenetic clades of Trichodesmium

Trichodesmium spp. play key roles in global carbon and nitrogen budgets and thus defining what controls their productivity is important for understanding climate change. While iron availability has been shown to be an important chemical factor for controlling both growth and nitrogen fixation rates in Trichodesmium , all culture experiments to date have focused solely on representatives from one clade of Trichodesmium . Genomic sequence analysis determined that the Trichodesmium erythraeum (IMS101) genome contains many of the archetypical genes involved in the prokaryotic iron stress response. Focusing on three of these genes, isiB , idiA and feoB , we found that all three showed an iron stress response in axenic T. erythraeum (IMS101), and their sequences were well conserved across four species in our Trichodesmium culture collection [consisting of two T. erythraeum strains (IMS101 and GBRTRLI101), two Trichodesmium tenue strains (Z-1 and H9-4), Trichodesmium thiebautii and Trichodesmium spiralis ]. With clade-specific quantitative PCR (qPCR) primers for one of these genes, isiB , we found that high isiB expression at low Fe levels corresponded to specific reductions in N₂ fixation rates in both major phylogenetic clades of Trichodesmium (the T. erythraeum clade and T. tenue clade). With regard to the two clades, the most significant difference determined was temperature optima, while more subtle differences in growth, N₂ fixation rate and gene expression responses to Fe stress were also observed. However the apparent conservation of the Fe stress response in the Trichodesmium genus suggests that it is an important adaptation for their niche in the oligotrophic ocean.     

CYTOMORPHOLOGICAL CHARACTERIZATION OF THE PLANKTONIC DIAZOTROPHIC CYANOBACTERIA TRICHODESMIUM SPP. FROM THE INDIAN OCEAN AND CARIBBEAN AND SARGASSO SEAS1

Trichodesmium Ehrenberg species were collected in the Caribbean Sea, Sargasso Sea, and coastal areas of Tanzania (Indian Ocean). The specimens were divided into five species on the basis of morphometric characters such as cell dimensions and colony formation: T. tenue Wille, T. erythraeum Ehrenberg, T. thiebautii Gomont, T. hildebrandtii Gomont, and T. contortum Wille. In addition, Trichodesmium sp., a spherical colony of uncertain taxonomic position was examined. The cell structure of each species was investigated by means of light, scanning electron, and transvnission electron microscopy. Particular attention was paid to the presence and ultrastructural arrangement of gas vacuoles and glycogen fiber clusters (GFCs). This resulted in identification of two major groups of species: 1) T. tenue, Trichodesmium sp. with spherical-shaped colonies, and T. erythraeum with GFCs and more or less localized gas vacuoles; and 2) T. thiebautii, T. hildebrandtii, and T. contortum lacking GFCs and with gas vacuoles spread at random. The species within each group were further characterized with respect to the dimension of the gas vesicles, cylindrical bodies, scroll bodies, and a new cellular inclusion body, Differences in colony formation and cell dimensions correlated with specific ultrastructural characters in five of the six forms. This is the first ultrastructural study comparing different forms of Trichodesmium sampled at geographically remote areas and shows that one species appears identical regardless of the sampling site. Some of the species had not been investigated earlier, and probably more species are to be identified and analyzed.     

Molecular and morphological criteria for revision of the genus Microcoleus (Oscillatoriales,Cyanobacteria)

Ninety‐two strains of Microcoleus vaginatus (=nomenclatural‐type species of the genus Microcoleus Desmazières ex Gomont) and Phormidium autumnale Trevisan ex Gomont from a wide diversity of regions and biotopes were examined using a combination of morphological and molecular methods. Phylogenies based on the 16S rDNA and 16S‐23S ITS (partial) demonstrated that the 92 strains, together with a number of strains in GenBank, were members of a highly supported monophyletic clade of strains (Bayesian posterior probability = 1.0) distant from the species‐cluster containing the generitype of Phormidium. Similarity of the 16S rRNA gene exceeded 95.5% among all members of the Microcoleus clade, but was less than 95% between any Microcoleus strains and species outside of the clade (e.g., Phormidium sensu stricto). These findings, which are in agreement with earlier studies on these taxa, necessitate the revision of Microcoleus to include P. autumnale. Furthermore, the cluster of Phormidium species in the P. autumnale group (known as Group VII) must be moved into Microcoleus as well, and these nomenclatural transfers are included in this study. The main diacritical characters defining Microcoleus are related to the cytomorphology of trichomes, including: narrowed trichome ends, calyptra, cells shorter than wide up to more or less isodiametric, and facultative presence of sheaths. The majority of species are 4–10 μm in diameter. The possession of multiple trichomes in a common sheath is present facultatively in many but not all species.     

COLEOFASCICULUS GEN. NOV. (CYANOBACTERIA): MORPHOLOGICAL AND MOLECULAR CRITERIA FOR REVISION OF THE GENUS MICROCOLEUS GOMONT1

Species currently classified within the cyanobacterial genus Microcoleus were determined to fall into two distinct clades in a 16S rDNA phylogeny, one containing taxa within the Oscillatoriaceae, the other containing taxa within the Phormidiaceae. The two lineages were confirmed in an analysis of the 16S–23S internal transcribed spacer (ITS) region sequences and secondary structures. The type species for Microcoleus is M. vaginatus Gomont, and this taxon belongs in the Oscillatoriaceae. Consequently, Microcoleus taxa in the Phormidiaceae must be placed in separate genera, and we propose the new genus Coleofasciculus to contain marine taxa currently placed in Microcoleus. The type species for Coleofasciculus is the well‐studied and widespread marine mat‐forming species Microcoleus chthonoplastes (Mert.) Zanardini ex Gomont. Other characters separating the two families include type of cell division and thylakoid structure.     

Phylogenies from genetic and morphological characters do not support a revision of Gigasporaceae (Glomeromycota) into four families and five genera

The family Gigasporaceae consisted of the two genera Gigaspora and Scutellospora when first erected. In a recent revision of this classification, Scutellospora was divided into three families and four genera based on two main lines of evidence: (1) phylogenetic patterns of coevolving small and large rRNA genes and (2) morphology of spore germination shields. The rRNA trees were assumed to accurately reflect species evolution, and shield characters were selected because they correlated with gene trees. These characters then were used selectively to support gene trees and validate the classification. To test this new classification, a phylogenetic tree was reconstructed from concatenated 25S rRNA and β-tubulin gene sequences using 35% of known species in Gigasporaceae. A tree also was reconstructed from 23 morphological characters represented in 71% of known species. Results from both datasets showed that the revised classification was untenable. The classification also failed to accurately represent sister group relationships amongst higher taxa. Only two clades were fully resolved and congruent among datasets: Gigaspora and Racocetra (a clade consisting of species with spores having one inner germinal wall). Other clades were unresolved, which was attributed in part to undersampling of species. Topology of the morphology-based phylogeny was incongruent with gene evolution. Five shield characters were reduced to three, of which two were phylogenetically uninformative because they were homoplastic. Therefore, most taxa erected in the new classification are rejected. The classification is revised to restore the family Gigasporaceae, within which are the three genera Gigaspora, Racocetra, and Scutellospora. This classification does not reflect strict topology of either gene or morphological evolution. Further revisions must await sampling of additional characters and taxa to better ascertain congruence between datasets and infer a more accurate phylogeny of this important group of fungi.     

A COMBINED 18S rDNA AND rbcL PHYLOGENETIC ANALYSIS OF CHLOROMONAS AND CHLAMYDOMONAS (CHLOROPHYCEAE,VOLVOCALES) EMPHASIZING SNOW AND OTHER COLD‐TEMPERATURE HABITATS1

An extensive phylogenetic analysis of the biflagellate genera, Chlamydomonas Ehrenberg and Chloromonas Gobi emend. Wille, was undertaken using 18S rDNA and rbcL gene sequence analysis. Emphasis was placed on 21 cold‐tolerant taxa of which 10 are from snow. These taxa occurred in four distinct clades each in the 18S rDNA and rbcL phylogenies, and when taken together suggest at least five distinct origins in cold habitats. Most of these taxa occur in a single clade (A), and all snow species occurred in this clade. In the rbcL and combined rbcL–18S rDNA analyses, the snow taxa fell into three groups. Two groups occurred in subclade 1: Chlamydomonas augustae Skuja CU, Chlamydomonas augustae UTEX, and Chlamydomonas sp.‐A and Chloromonas clathrata Korshikov, Chloromonas rosae Ettl CU, and Chloromonas rosae v. psychrophila var. nov. The third snow group, subclade 2, included three species with unique cell divisions, Chloromonas brevispina (Fritsch) Hoham, Roemer et Mullet, Chloromonas pichinchae (Lagerheim) Wille, and Chloromonas sp.‐D, and the basal Chloromonas nivalis (Chodat) Hoham et Mullet with normal cell divisions. This suggests that the snow habitat has been colonized at least twice and possibly three times in the history of these biflagellates. In the 18S rDNA tree, one cold‐tolerant Chloromonas species fell outside clade A: Chloromonas subdivisa (Pascher et Jahoda) Gerloff et Ettl. In the rbcL tree, three cold‐tolerant Chloromonas species fell outside clade A: Chloromonas subdivisa, Chloromonas sp.‐ANT1, and Chloromonas sp.‐ANT3. These results support previous findings that pyrenoids have been gained and lost several times within this complex.     

Phylogeny of the spider genus Ixchela Huber, 2000 (Araneae: Pholcidae) based on morphological and molecular evidence (CO1 and 16S), with a hypothesized diversification in the Pleistocene

The genus Ixchela Huber is composed of 20 species distributed from north‐eastern Mexico to Central America, including the five new species described here from Mexico: I xchela azteca sp. nov. , I xchela jalisco sp. nov. , I xchela mendozai sp. nov. , I xchela purepecha sp. nov. and I xchela tlayuda sp. nov. We test the monophyly and investigate the phylogenetic relationships among species of the genus Ixchela using morphological and molecular data. Parsimony (PA) analysis of 24 taxa and 40 morphological characters with equal and implied weights supported the monophyly of Ixchela with eight morphological synapomorphies. The PA analyses with equal and implied weights, and separate Bayesian inference (BI) analyses for the CO1 gene (506 characters), concatenated gene fragments CO1 + 16S (885 characters), morphology + CO1 (546 characters) and the combined evidence data set (morphology + CO1 + 16S) (925 characters) support the monophyly of Ixchela. Our preferred topology shows two large clades; clade 1 has a natural distribution in the Mesoamerican biotic component, whereas clade 2 predominates in the Mexican Montane biotic component. The genus Ixchela diverged in the late Miocene, and the divergence between the internal clades in the genus occurred in the late Pliocene; by contrast, most of the speciation events seem to have occurred mainly during the Pleistocene, where climatic changes brought on by repeated glaciations played an important role in the diversification of the genus. © 2015 The Linnean Society of London     

Phylogenetic relationships in the millipede family Julidae

A phylogenetic analysis of 40 species (22 genera) of the Palaearctic millipede family Julidae was made based on partial sequences of the mitochondrial 16S rRNA (16S) gene and the nuclear 28S rRNA (28S) gene, respectively. The two data sets (16S rDNA and 28S rDNA) were analysed individually and in combination using direct optimization as implemented in POY. The 16S rDNA and the 28S rDNA sequences vary from 410 to 449 bp and from 467 to 525 bp in length, respectively. All searches were performed under six different gap opening costs, an extension gap cost of 1, and a substitution cost of 2. Incongruence length difference values were used to select the preferred tree. The order Julida was recovered as monophyletic under all weight sets. The family Julidae was recovered as monophyletic except under one weight set where the genus Nepalmatoiulus is sister to all other Julida. Within Julidae, a clade of Paectophyllini + Calyptophyllini is sister to all others on the preferred tree but this relationship is not robust. A hitherto unrecognized clade of (South) east Asian genera (Anaulaciulus and Nepalmatoiulus) was recovered under five weight sets. Another “new” robust clade (Oncoiulini + Schizophyllini) is congruent with a hitherto unrecognized complex morphological character. Further clades recovered within the Julidae partly conflict with the accepted classification, which is only to a limited extent based on phylogenetic arguments.     

Phylogeny of Chaetonotidae and other Paucitubulatina (Gastrotricha: Chaetonotida) and the colonization of aquatic ecosystems

Kånneby, T., Todaro, M. A., Jondelius, U. (2012). Phylogeny of Chaetonotidae and other Paucitubulatina (Gastrotricha: Chaetonotida) and the colonization of aquatic ecosystems. —Zoologica Scripta, 42, 88–105. Chaetonotidae is the largest family within Gastrotricha with almost 400 nominal species represented in both freshwater and marine habitats. The group is probably non‐monophyletic and suffers from a troubled taxonomy. Current classification is to a great extent based on shape and distribution of cuticular structures, characters that are highly variable. We present the most densely sampled molecular study so far where 17 of the 31 genera belonging to Chaetonotida are represented. Bayesian and maximum likelihood approaches based on 18S rDNA, 28S rDNA and COI mtDNA are used to reconstruct relationships within Chaetonotidae. The use of cuticular structures for supra‐specific classification within the group is evaluated and the question of dispersal between marine and freshwater habitats is addressed. Moreover, the subgeneric classification of Chaetonotus is tested in a phylogenetic context. Our results show high support for a clade containing Dasydytidae nested within Chaetonotidae. Within this clade, only three genera are monophyletic following current classification. Genera containing both marine and freshwater species never form monophyletic clades and group with other species according to habitat. Marine members of Aspidiophorus appear to be the sister group of all other Chaetonotidae and Dasydytidae, indicating a marine origin of the clade. Halichaetonotus and marine Heterolepidoderma form a monophyletic group in a sister group relationship to freshwater species, pointing towards a secondary invasion of marine environments of these taxa. Our study highlights the problems of current classification based on cuticular structures, characters that show homoplasy for deeper relationships.     

Morphological and phylogenetic analyses of Pythium species in South Africa

The genus Pythium is important in agriculture, since it contains many plant pathogenic species, as well as species that can promote plant growth and some that have biocontrol potential. In South Africa, very little is known about the diversity of Pythium species within agricultural soil, irrigation and hydroponic systems. Therefore, the aim of the study was to characterise a selection of 85 Pythium isolates collected in South Africa from 1991 through to 2007. The isolates were characterised morphologically as well as through sequence and phylogenetic analyses of the internal transcribed spacer regions (ITS) and the 5.8S gene of the nuclear ribosomal DNA. Phylogenetic analyses showed that the isolates represented ten of the 11 published Pythium clades [Lévesque & De Cock, 2004. Molecular phylogeny and taxonomy of the genus Pythium. Mycological Research 108: 1363–1383]. Characterisation of isolates in clade D and J suggested that the phylogenetic concept of Pythium acanthicum and Pythium perplexum respectively, needs further investigation in order to enable reliable species identification within these clades. Our phylogenetic analyses of Pythium species in clade B also showed that species with globose sporangia group basal within this clade, and are not dispersed within the clade as previously reported. The 85 South African isolates represented 34 known species, of which 20 species have not been reported previously in South Africa. Additionally, three isolates (PPRI 8428, 8300 and 8418) were identified that may each represent putative new species, Pythium sp. WJB-1 to WJB-3.     

Igniting taxonomic curiosity: The amazing story of Amazonocrinis with the description of a new genus Ahomia gen. nov. and novel species of Ahomia,Amazonocrinis, and Dendronalium from the biodiversity-rich northeast region of India

Five cyanobacterial strains exhibiting Nostoc-like morphology were sampled from the biodiversity hotspots of the northeast region of India and characterized using a polyphasic approach. Molecular and phylogenetic analysis using the 16S rRNA gene indicated that the strains belonged to the genera Amazonocrinis and Dendronalium. In the present investigation, the 16S rRNA gene phylogeny clearly demarcated two separate clades of Amazonocrinis. The strain MEG8-PS clustered along with Amazonocrinis nigriterrae CENA67, which is the type strain of the genus. The other three strains ASM11-PS, RAN-4C-PS, and NP-KLS-5A-PS clustered in a different clade that was phylogenetically distinct from the Amazonocrinis sensu stricto clade. Interestingly, while the 16S rRNA gene phylogeny exhibited two separate clusters, the 16S–23S ITS region analysis did not provide strong support for the phylogenetic observation. Subsequent analyses raised questions regarding the resolving power of the 16S–23S ITS region at the genera level and the associated complexities in cyanobacterial taxonomy. Through this study, we describe a novel genus Ahomia to accommodate the members clustering outside the Amazonocrinis sensu stricto clade. In addition, we describe five novel species, Ahomia kamrupensis, Ahomia purpurea, Ahomia soli, Amazonocrinis meghalayensis, and Dendronalium spirale, in accordance with the International Code of Nomenclature for algae, fungi, and plants (ICN). Apart from further enriching the genera Amazonocrinis and Dendronalium, the current study helps to resolve the taxonomic complexities revolving around the genus Amazonocrinis and aims to attract researchers to the continued exploration of the tropical and subtropical cyanobacteria for interesting taxa and lineages.     

Genetic diversity and phylogenetic analyses of ixodid ticks infesting cattle in northeast Thailand: the discovery of Rhipicephalus microplus clade C and the rarely detected R. haemaphysaloides

In total, 160 ticks infesting cattle in the northeast region of Thailand were collected and used for molecular investigation. Three tick species—Rhipicephalus microplus Canestrini, Rhipicephalus haemaphysaloides Supino and Haemaphysalis bispinosa Neumann—were identified based on morphology and DNA sequences of mitochondrial cytochrome c oxidase subunit 1 (CO1) and 16S ribosomal RNA (16S rRNA). In total, 26 and seven unique haplotypes of the CO1 and 16S rRNA genes, respectively, were recovered. Phylogenetic analysis using the CO1 sequence revealed that the R. microplus from northeastern Thailand were grouped into the previously described clades A and C, whereas the 16S rRNA phylogenetic tree assigned all isolates of R. microplus from Northeast Thailand into the previously described clade B. Clade C of the CO1 phylogenetic tree is a new genetic assemblage recently discovered from India and Malaysia, which has now been detected in our study. The haplotype network also demonstrated that R. microplus is divided into two haplogroups corresponding to the assemblage of the CO1 phylogenetic tree. Our findings strongly support the previous genetic assemblage classification and evidence that R. microplus from Northeast Thailand is a species complex comprising at least two genetic assemblages, i.e., clades A and C. However, further investigation is needed and should involve more comprehensive genetic and morphological analyses and cover a larger part of their distributional range throughout Southeast Asia.

     

GENOTYPIC RELATIONSHIPS IN TRICHODESMIUM (CYANOPHYCEAE) BASED ON nifH SEQUENCE COMPARISONS1

The DNA sequence of a fragment of nifH was compared to natural populations of the marine cyanobacteria Trichodesmium thiebautii and T. erythraeum from the Caribbean Sea and the unialgal culture Trichodesmium sp. NIBB 1067, which was isolated from the Kuroshio waters (Japan). Through replication Of amplification, cloning, and sequencing, four nucleotides in a 359-bp fragment were identified that were identical in sequence to Trichodesmium sp. NIBB 1067 and natural populations of T. erythraeum but were distinctly different in sequence from T. thiebautii. The data indicate that Trichodesmium sp. NIBB 1067 is more closely related to T. erythraeum than to T. thiebautii.     

Comparison of Leptospira species isolated from environmental water and soil in Japan

Leptospira was isolated from environmental water in central Japan using selective medium comprising five antibiotics, namely sulfamethoxazole, trimethoprim, amphotericin B, fosfomycin, and 5‐fluorouracil. Of 100 water samples 57 (57%) were culture‐positive and 50 pure cultures were isolated. Of the 50 cultures isolated from water 48 were classified into a saprophytic clade on the basis of 16S ribosomal RNA gene sequences. However, it was previously reported that isolates from soil in Japan belonged to pathogenic, intermediate, and saprophytic clades, the current findings suggest less diversity of Leptospira species in environmental water than that in soil in Japan.     

Systematics of Cladophora spp. (Chlorophyta) from North Carolina,USA, based upon morphology and DNA sequence data with a description of Cladophora subtilissima sp. nov.

Identification of Cladophora species is challenging due to conservation of gross morphology, few discrete autapomorphies, and environmental influences on morphology. Twelve species of marine Cladophora were reported from North Carolina waters. Cladophora specimens were collected from inshore and offshore marine waters for DNA sequence and morphological analyses. The nuclear‐encoded rRNA internal transcribed spacer regions (ITS) were sequenced for 105 specimens and used in molecular assisted identification. The ITS1 and ITS2 region was highly variable, and sequences were sorted into ITS Sets of Alignable Sequences (SASs). Sequencing of short hyper‐variable ITS1 sections from Cladophora type specimens was used to positively identify species represented by SASs when the types were made available. Secondary structures for the ITS1 locus were also predicted for each specimen and compared to predicted structures from Cladophora sequences available in GenBank. Nine ITS SASs were identified and representative specimens chosen for phylogenetic analyses of 18S and 28S rRNA gene sequences to reveal relationships with other Cladophora species. Phylogenetic analyses indicated that marine Cladophorales were polyphyletic and separated into two clades, the Cladophora clade and the “Siphonocladales” clade. Morphological analyses were performed to assess the consistency of character states within species, and complement the DNA sequence analyses. These analyses revealed intra‐ and interspecific character state variation, and that combined molecular and morphological analyses were required for the identification of species. One new report, Cladophora dotyana, and one new species Cladophora subtilissima sp. nov., were revealed, and increased the biodiversity of North Carolina marine Cladophora to 14 species.