Cryptic Speciation in the Genus Cryptoglena (Euglenaceae) Revealed by Nuclear and Plastid SSU and LSU rRNA Gene

The photosynthetic euglenoid genus Cryptoglena is differentiated from other euglenoid genera by having a longitudinal sulcus, one chloroplast, two large trough‐shaped paramylon plates positioned between the chloroplast and pellicle, and lack of metaboly. The genus contains only two species. To understand genetic diversity and taxonomy of Cryptoglena species, we analyzed molecular and morphological data from 25 strains. A combined data set of nuclear SSU and LSU and plastid SSU and LSU rRNA genes was analyzed using Bayesian, maximum likelihood, maximum parsimony, and distance (neighbor joining) methods. Although morphological data of all strains showed no significant species‐specific pattern, molecular data segregated the taxa into five clades, two of which represented previously known species: C. skujae and C. pigra, and three of which were designated as the new species, C. soropigra, C. similis, and C. longisulca. Each species had unique molecular signatures that could be found in the plastid SSU rRNA Helix P23_1 and LSU rRNA H2 domain. The genetic similarity of intraspecies based on nr SSU rDNA ranged from 97.8% to 100% and interspecies ranged from 95.3% to 98.9%. Therefore, we propose three new species based on specific molecular signatures and gene divergence of the nr SSU rDNA sequences.     

Deep segregation in the open ocean: Macaronesia as an evolutionary hotspot for low dispersal marine invertebrates

Diversification and speciation of terrestrial organisms are anticipated in oceanic islands such as Macaronesia, a group of Atlantic islands that have remained unconnected to continental landmasses. Hitherto, the diversification of marine organisms in oceanic islands, especially those with low vagility, has received little direct empirical analysis using molecular markers. Here, we focus on such a case study, through applying a multilocus molecular approach to investigate the diversity and evolution of a group that lacks a planktonic larval stage, the isopod genus Dynamene, in Macaronesia and Northeast Atlantic. Sequences of two mitochondrial (cytochrome c oxidase subunit I and 16S rRNA) and two nuclear (18S rRNA and 28S rRNA) loci were obtained from specimens of Dynamene edwardsi (Lucas, 1849), Dynamene magnitorata Holdich, 1968 and Dynamene bidentata (Adams, 1800) collected along the Northeast Atlantic and Macaronesia. Although no major phylogeographic structure was detected in D. bidentata and D. magnitorata, from five to nine deeply divergent lineages were evident within D. edwardsi. The divergent lineages displayed genetic distances comparable to those found among established species of peracarids. D. edwardsi exhibits a long, rich and complex phylogeographic history in Macaronesia, where the geodynamics of the islands possibly associated with founder effects and subsequent lack of gene flow among populations confounds patterns based on geographic proximity of targeted populations. Our findings collectively suggest a much larger role of oceanic islands in the diversification of marine invertebrates than previously anticipated. The work provides insights into the origins and dynamics of ongoing geographic segregation and associated deep divergence among sister evolutionary lineages in Macaronesia.     

Development and preliminary application of a triplex real‐time polymerase chain reaction assay for evaluating predation on three planthoppers in a rice ecosystem

A multiplex real‐time quantitative polymerase chain reaction (PCR) assay was developed to simultaneously detect the DNA of three rice planthoppers, that is, Sogatella furcifera (Horváth) (white‐backed planthopper), Nilaparvata lugens (Stål) (brown planthopper) and Laodelphax striatellus (Fallén) (small brown planthopper), in the gut of their predators. The sets of primers and ALLGlo probes were targeted to the regions of internal transcribed spacer 2 (ITS2) genes in nuclear ribosomal DNA (rDNA). The sensitivity, specificity and interference test for the multiplex real‐time quantitative PCR assay were analysed. The assay's detection limits were 100, 1000 and 100 copies for the white‐backed planthopper, the brown planthopper and the small brown planthopper, respectively. The specificity tests showed no cross‐reactivity with genomic DNA from 30 other dominant herbivores, saprophagous insects and predators from rice ecosystem for each planthopper species. The assay was used in a preliminary study of predation events on the three planthoppers by three major spiders viz., Pardosa pseudoannulata (Bösenberg et Strand), Ummeliata insecticeps (Bösenberg et Strand) and Tetragnatha maxillosa Thorell which each differ in their preferred microhabitat as well as their predatory habits in rice field, and the results showed their predation on each planthopper species could be well evaluated using this method. Therefore, the multiplex real‐time quantitative PCR assay provides a new tool to study the mechanisms of prey shifting and natural regulation of the three rice planthoppers by generalist predators in rice ecosystem.     

Seasonal dietary shifts enhance parasite transmission to lake salmonids during ice cover

Changes in abiotic and biotic factors between seasons in subarctic lake systems are often profound, potentially affecting the community structure and population dynamics of parasites over the annual cycle. However, few winter studies exist and interactions between fish hosts and their parasites are typically confined to snapshot studies restricted to the summer season whereas host‐parasite dynamics during the ice‐covered period rarely have been explored. The present study addresses seasonal patterns in the infections of intestinal parasites and their association with the diet of sympatric living Arctic charr (Salvelinus alpinus) and brown trout (Salmo trutta) in Lake Takvatn, a subarctic lake in northern Norway. In total, 354 Arctic charr and 203 brown trout were sampled from the littoral habitat between June 2017 and May 2018. Six trophically transmitted intestinal parasite taxa were identified and quantified, and their seasonal variations were contrasted with dietary information from both stomachs and intestines of the fish. The winter period proved to be an important transmission window for parasites, with increased prevalence and intensity of amphipod‐transmitted parasites in Arctic charr and parasites transmitted through fish prey in brown trout. In Arctic charr, seasonal patterns in parasite infections resulted mainly from temporal changes in diet toward amphipods, whereas host body size and the utilization of fish prey were the main drivers in brown trout. The overall dynamics in the community structure of parasites chiefly mirrored the seasonal dietary shifts of their fish hosts.     

First Report of Leaf Blight Disease Caused by Fusarium verticillioides (Teleomorph Gibberella moniliformis) in Kentucky Bluegrass

Leaf blight symptom caused by Gibberella moniliformis was observed in the seedlings of Kentucky bluegrass (cv. Perfection) in Jinju, Korea, in January 2013. The symptoms first appeared as brown to yellow lesions on leaves, which later became white and bleached. The pathogen was isolated from turfgrass leaves and identified as G. moniliformis by using the internal transcribed spacer (ITS)1 and ITS4 sequences of rRNA. G. moniliformis was confirmed by a pathogenicity test, and the causal agent was reisolated from infected turfgrass.     

Future warming and acidification effects on anti‐fouling and anti‐herbivory traits of the brown alga Fucus vesiculosus (Phaeophyceae)

Human‐induced ocean warming and acidification have received increasing attention over the past decade and are considered to have substantial consequences for a broad range of marine species and their interactions. Understanding how these interactions shift in response to climate change is particularly important with regard to foundation species, such as the brown alga Fucus vesiculosus. This macroalga represents the dominant habitat former on coastal rocky substrata of the Baltic Sea, fulfilling functions essential for the entire benthic community. Its ability to withstand extensive fouling and herbivory regulates the associated community and ecosystem dynamics. This study tested the interactive effects of future warming, acidification, and seasonality on the interactions of a marine macroalga with potential foulers and consumers. F. vesiculosus rockweeds were exposed to different combinations of conditions predicted regionally for the year 2100 (+?5°C, +?700 μatm CO₂) using multifactorial long‐term experiments in novel outdoor benthic mesocosms (“Benthocosms”) over 9–12‐week periods in four seasons. Possible shifts in the macroalgal susceptibility to fouling and consumption were tested using consecutive bioassays. Algal susceptibility to fouling and grazing varied substantially among seasons and between treatments. In all seasons, warming predominantly affected anti‐fouling and anti‐herbivory interactions while acidification had a subtle nonsignificant influence. Interestingly, anti‐microfouling activity was highest during winter under warming, while anti‐macrofouling and anti‐herbivory activities were highest in the summer under warming. These contrasting findings indicate that seasonal changes in anti‐fouling and anti‐herbivory traits may interact with ocean warming in altering F. vesiculosus community composition in the future.     

Phylogenetic Investigations on Ten Genera of Tintinnid Ciliates (Ciliophora: Spirotrichea: Tintinnida), Based on Small Subunit Ribosomal RNA Gene Sequences

Tintinnida is a diverse taxon that accommodates over 1,500 morphospecies, which is an important component of marine planktonic food webs. However, evolutionary relationships of tintinnids are poorly known because molecular data of most groups within this order are lacking. In our study, the small subunit (SSU) rRNA genes representing 10 genera, 5 families of Tintinnida were sequenced, including the first SSU rRNA gene sequences for Coxliella, Dadayiella, Epiplocyloides, and Protorhabdonella, and phylogenetic trees were constructed to assess their intergeneric relationships. Phylogenies inferred from different methods showed that (1) Three newly sequenced Eutintinnus species fell into Eutintinnus clade forming a sister group to the clade containing Amphorides, Steenstrupiella, Amphorellopsis, and Salpingella; (2) Surprisingly, the genetic distances between Amphorides amphora and Amphorellopsis acuta population 1 was even smaller than that between the two populations of Amphorellopsis acuta, casting doubt on the validity of Amphorides and Amphorellopsis as presently defined; (3) The SSU rRNA sequences of Dadayiella ganymedes and Parundella aculeata were almost identical. Therefore, Parundella ganymedes novel combination is proposed; (4) Coxliella, which is currently assigned within Metacylididae, branched instead with some Tintinnopsis species. Furthermore, the validation of Coxliella, which was considered to be a “questionable” genus, was confirmed based on evidences from morphology, ecology, and molecular data; (5) Protorhabdonella and Rhabdonella showed rather low intergeneric distance and grouped together with strong support suggesting that Rhabdonellidae is a well‐defined taxon; and (6) Epiplocyloides branched with species in Cyttarocylididae indicating their close relationship.     

Effect of Zinc on the Development of Brown Spot in Rice

Brown spot, caused by the fungus Bipolaris oryzae, is one of the most destructive diseases of rice. This study investigated the effect of zinc rates on the development of brown spot in rice. Rice plants (cv. ‘Metica‐1′) were grown in hydroponic culture amended with Zn rates (applied as ZnSO₄^.7H₂O) of 0, 0.5, 1, 2 and 4 μm and inoculated with B. oryzae. The foliar concentration of Zn was determined. Leaf samples were assessed for disease severity, and then, area under brown spot progress curve (AUBSPC) was calculated. The relationship between Zn concentrations on leaf tissues and the rates of this micronutrient was best described by a positive linear regression model, while the relationship between the Zn rates and the AUBSPC was best described with a positive quadratic regression model. The correlation between Zn concentrations on leaf tissues and AUBSPC was positive and significant (r = 0.68, P < 0.05). The results from this study showed that high foliar concentration of Zn was associated with increasing rice susceptibility to brown spot.     

Existence of Novel Phylotypes of Nitrite-Dependent Anaerobic Methane-Oxidizing Bacteria in Surface and Subsurface Sediments of the South China Sea

Nitrite-dependent anaerobic methane oxidation (n-damo) is a recently discovered new microbial process performed by the Candidatus Methylomirabilis oxyfera with an unusual intra-aerobic pathway, but there is no report about n-damo bacteria in marine environments. M. oxyfera-like sequences were successfully retrieved for the first time from both surface and subsurface ocean sediments of the South China Sea (SCS) using both 16S rRNA and pmoA genes as biomarkers and PCR amplification in this study. The majority of M. oxyfera-like 16S rRNA gene-based PCR amplified sequences from the SCS sediments formed a new group distinctively different from those detected in freshwater habitats and the information is consistent phylogenetically with those obtained from the pmoA gene. This study showed the existence of n-damo in ocean sediments and suggests that marine sediments harbor n-damo phylotypes different from those in the freshwater. This finding here expands our understanding on the distribution of n-damo bacteria to marine ecosystem and implies their potential contribution to the marine C and N cycling.     

First Report of Anthracnose of Ledebouriella seseloides Caused by Colletotrichum gloeosporioides in Korea

In 2012, dark brown spots were observed on leaves of Ledebouriella seseloides (Fang Feng) in several research plots located at the Goseong Agricultural Research Extension services in Gyeongam Province, Republic of Korea. A fungus was isolated from the infected plants which produced pink‐coloured spores in mucilage on PDA and conidial morphology suggested that the causal agent was Colletotrichum gloeosporioides. Internal transcribed spacer sequences of the pathogen showed 99% identity to those of C. gloeosporioides. Pathogenicity of the isolate was proved by Koch's postulates. This is the first report of anthracnose in L. seseloides caused by C. gloeosporioides.     

Morphological and genetic diversity of Beaufort Sea diatoms with high contributions from the Chaetoceros neogracilis species complex

Seventy‐five diatom strains isolated from the Beaufort Sea (Canadian Arctic) in the summer of 2009 were characterized by light and electron microscopy (SEM and TEM), as well as 18S and 28S rRNA gene sequencing. These strains group into 20 genotypes and 17 morphotypes and are affiliated with the genera Arcocellulus, Attheya, Chaetoceros, Cylindrotheca, Eucampia, Nitzschia, Porosira, Pseudo‐nitzschia, Shionodiscus, Thalassiosira, and Synedropsis. Most of the species have a distribution confined to the northern/polar area. Chaetoceros neogracilis and Chaetoceros gelidus were the most represented taxa. Strains of C. neogracilis were morphologically similar and shared identical 18S rRNA gene sequences, but belonged to four distinct genetic clades based on 28S rRNA, ITS‐1 and ITS‐2 phylogenies. Secondary structure prediction revealed that these four clades differ in hemi‐compensatory base changes (HCBCs) in paired positions of the ITS‐2, suggesting their inability to interbreed. Reproductively isolated C. neogracilis genotypes can thus co‐occur in summer phytoplankton communities in the Beaufort Sea. C. neogracilis generally occurred as single cells but also formed short colonies. It is phylogenetically distinct from an Antarctic species, erroneously identified in some previous studies as C. neogracilis, but named here as Chaetoceros sp. This work provides taxonomically validated sequences for 20 Arctic diatom taxa, which will facilitate future metabarcoding studies on phytoplankton in this region.     

Suppression of Rust and Brown Eye Spot Diseases on Coffee by Phosphites and By‐products of Coffee and Citrus Industries

The rust and brown eye spot, caused by Hemileia vastatrix and Cercospora coffeicola, respectively, are the most important fungal diseases on coffee in South America. Their management is mainly by chemical treatment, and there is no genetic resistance to brown eye spot known so far. Considering the need for developing alternative products for their control, the goal of this work was to evaluate the effects of phosphites and by‐products of coffee and citrus industries on rust and brown eye spot. Formulations of coffee and citrus industry by‐products, phosphites and their combination with fungicide were evaluated in field experiments, and their effect on fungal urediniospores and conidia was evaluated in vitro. In the field, treatments were applied individually or in combination and the in vitro assays were performed with manganese phosphite (Reforce Mn), potassium phosphite and citrus industry by‐product (Fortaleza), copper phosphite and coffee industry by‐product (Fitoforce Full), and fungicide. The severity and incidence of rust and brown eye spot on coffee leaves, yield, and leaf retention were evaluated in the field. Percentage of spore germination was evaluated in vitro for both fungi, whereas mycelial growth was evaluated for C. coffeicola only. The treatments Fortaleza, Reforce Mn and Fitoforce Full suppressed both diseases with a reduction in defoliation. In the year 2012, the plants treated with Reforce Mn and Reforce Mn + Fortaleza showed a yield increase of 72 and 88%, respectively, which was similar to the results shown by the fungicide treatment. In vitro inhibition of germination of H. vastatrix urediniospores and of C. coffeicola conidia was observed and suggests that the products exert some toxic effects to both fungi. Finally, the results observed indicate that the combined use of by‐products of plant‐processing industries and phosphites is an alternative and can be added efficiently to the management of coffee diseases.     

Species delimitation and phylogeographic analyses in the Ectocarpus subgroup siliculosi (Ectocarpales,Phaeophyceae)

The genus Ectocarpus (Ectocarpales, Phaeophyceae) contains filamentous algae widely distributed in marine and estuarine habitats of temperate regions in both hemispheres. While E. siliculosus has become a model organism for genomics and genetics of the brown macroalgae, accurate species delineation, distribution patterns and diversity for the genus Ectocarpus remain problematic. In this study, we used three independent species delimitation approaches to generate a robust species hypothesis for 729 Ectocarpus specimens collected mainly along the European and Chilean coasts. These approaches comprised phylogenetic reconstructions and two bioinformatics tools developed to objectively define species boundaries (General Mixed Yule Coalescence Method and Automatic Barcode Gap Discovery). Our analyses were based on DNA sequences of two loci: the mitochondrial cytochrome oxidase subunit 1 and the nuclear internal transcribed spacer 1 of the ribosomal DNA. Our analyses showed the presence of at least 15 cryptic species and suggest the existence of incomplete lineage sorting or introgression between five of them. These results suggested the possible existence of different levels of reproductive barriers within this species complex. We also detected differences among species in their phylogeographic patterns, range and depth distributions, which may suggest different biogeographic histories (e.g., endemic species or recent introductions).     

Morphology and Phylogeny of Two Species of Loxodes (Ciliophora,Karyorelictea), with Description of a New Subspecies,Loxodes striatus orientalis subsp. n.

The morphology and phylogeny of Loxodes vorax and L. striatus orientalis subsp. n. were investigated based on infraciliature and small subunit (SSU) rRNA gene sequence data. Loxodes striatus orientalis subsp. n. was separated from L. striatus striatus stat. n. by having fewer dikinetids in the intrabuccal kinety (35–55 vs. 50–70) and a variable number of macronuclei (2–4 vs. 2). In addition, the SSU rRNA gene sequence of the new subspecies differs in 13 and 11 nucleotides from that of two populations of the nominotypic subspecies. We also summarized the morphological differences between Loxodes and Remanella based on the data available. Phylogenetic analyses revealed that the genus Loxodes was monophyletic and nested within Remanella species. This study might, therefore, support the hypothesis that the freshwater genus Loxodes evolved from the marine genus Remanella.     

Neuraminidase Inhibitors from marine‐derived actinomycete Streptomyces seoulensis

Aims

This work was performed to characterize new secondary metabolites with neuraminidase (NA) inhibitory activity from marine actinomycete strains.

Methods and Results

An actinomycete strain IFB‐A01, capable of producing new NA inhibitors, was isolated from the gut of shrimp Penasus orientalis and identified as Streptomyces seoulensis according to its 16S rRNA sequence (over 99% homology with that of the standard strain). Repeated chromatography of the methanol extract of the solid‐substrate culture of S. seoulensis IFB‐A01 led to the isolation of streptoseolactone ( 1 ), and limazepines G ( 2 ) and H ( 3 ). The structures of 1 – 3 were determined by a combination of IR, ESI‐MS, 1D (¹H and ¹³C NMR, and DEPT) and 2D NMR experiments (HMQC, HMBC, ¹H‐¹H COSY and NOESY). Compounds 1 – 3 showed significant inhibition on NA in a dose‐dependent manner with IC₅₀ values of 3·92, 7·50 and 7·37 μmol l^?1, respectively.

Conclusions

This is the first report of two new ( 1 and 2 ) and known ( 3 , recovered as a natural product for the first time in the work) NA inhibitors from the marine‐derived actinomycete S. seoulensis IFB‐A01.

Significance and Impact of the Study

The three natural NA inhibitors maybe of value for the development of drug(s) necessitated for the treatment of viral infections.     

Isolation and characterization of fatty acid methyl ester (FAME)‐producing Streptomyces sp. S161 from sheep (Ovis aries) faeces

An actinomycete producing oil‐like mixtures was isolated and characterized. The strain was isolated from sheep faeces and identified as Streptomyces sp. S161 based on 16S rRNA gene sequence analysis. The strain showed cellulase and xylanase activities. The ¹H nuclear magnetic resonance (NMR) spectra of the mixtures showed that the mixtures were composed of fatty acid methyl esters (52·5), triglycerides (13·7) and monoglycerides (9·1) (mol.%). Based on the gas chromatography–mass spectrometry (GC‐MS) analysis, the fatty acid methyl esters were mainly composed of C14‐C16 long‐chain fatty acids. The results indicated that Streptomyces sp. S161 could produce fatty acid methyl esters (FAME) directly from starch. To our knowledge, this is the first isolated strain that can produce biodiesel (FAME) directly from starch.

Significance and Impact of the Study

Nowadays, production of biodiesel is based on plant oils, animal fats, algal oils and microbial oils. Lipid mostly consists of triacylglycerols (TAG), and conversion of these lipids into fatty acid short‐chain alcohol esters (methanol or ethanol) is the final step in biodiesel production. In this study, an oil‐producing Streptomyces strain was isolated from sheep faeces. The oil was composed of C₁₄‐C₁₆ long‐chain fatty acid methyl esters, triglycerides and monoglycerides. This is the first isolated strain‐producing biodiesel (FAME) directly from starch. Due to showing cellulase and xylanase activities, the strain would be helpful for converting renewable lignocellulose into biodiesel directly.     

Local parasite lineage sharing in temperate grassland birds provides clues about potential origins of Galapagos avian Plasmodium

Oceanic archipelagos are vulnerable to natural introduction of parasites via migratory birds. Our aim was to characterize the geographic origins of two Plasmodium parasite lineages detected in the Galapagos Islands and in North American breeding bobolinks (Dolichonyx oryzivorus) that regularly stop in Galapagos during migration to their South American overwintering sites. We used samples from a grassland breeding bird assemblage in Nebraska, United States, and parasite DNA sequences from the Galapagos Islands, Ecuador, to compare to global data in a DNA sequence registry. Homologous DNA sequences from parasites detected in bobolinks and more sedentary birds (e.g., brown‐headed cowbirds Molothrus ater, and other co‐occurring bird species resident on the North American breeding grounds) were compared to those recovered in previous studies from global sites. One parasite lineage that matched between Galapagos birds and the migratory bobolink, Plasmodium lineage B, was the most common lineage detected in the global MalAvi database, matching 49 sequences from unique host/site combinations, 41 of which were of South American origin. We did not detect lineage B in brown‐headed cowbirds. The other Galapagos‐bobolink match, Plasmodium lineage C, was identical to two other sequences from birds sampled in California. We detected a close variant of lineage C in brown‐headed cowbirds. Taken together, this pattern suggests that bobolinks became infected with lineage B on the South American end of their migratory range, and with lineage C on the North American breeding grounds. Overall, we detected more parasite lineages in bobolinks than in cowbirds. Galapagos Plasmodium had similar host breadth compared to the non‐Galapagos haemosporidian lineages detected in bobolinks, brown‐headed cowbirds, and other grassland species. This study highlights the utility of global haemosporidian data in the context of migratory bird–parasite connectivity. It is possible that migratory bobolinks bring parasites to the Galapagos and that these parasites originate from different biogeographic regions representing both their breeding and overwintering sites.     

Mutant swarms of a totivirus‐like entities are present in the red macroalga Chondrus crispus and have been partially transferred to the nuclear genome

Chondrus crispus Stackhouse (Gigartinales) is a red seaweed found on North Atlantic rocky shores. Electrophoresis of RNA extracts showed a prominent band with a size of around 6,000 bp. Sequencing of the band revealed several sequences with similarity to totiviruses, double‐stranded RNA viruses that normally infect fungi. This virus‐like entity was named C. crispus virus (CcV). It should probably be regarded as an extreme viral quasispecies or a mutant swarm since low identity (<65%) was found between sequences. Totiviruses typically code for two genes: one capsid gene (gag) and one RNA‐dependent RNA polymerase gene (pol) with a pseudoknot structure between the genes. Both the genes and the intergenic structures were found in the CcV sequences. A nonidentical gag gene was also found in the nuclear genome of C. crispus, with associated expressed sequence tags (EST) and upstream regulatory features. The gene was presumably horizontally transferred from the virus to the alga. Similar dsRNA bands were seen in extracts from different life cycle stages of C. crispus and from all geographic locations tested. In addition, similar bands were also observed in RNA extractions from other red algae; however, the significance of this apparently widespread phenomenon is unknown. Neither phenotype caused by the infection nor any virus particles or capsid proteins were identified; thus, the presence of viral particles has not been validated. These findings increase the known host range of totiviruses to include marine red algae.