Protist distribution in the Western Fram Strait in summer 2010 based on 454‐pyrosequencing of 18S rDNA

In this study, we present the first comprehensive analyses of the diversity and distribution of marine protist (micro‐, nano‐, and picoeukaryotes) in the Western Fram Strait, using 454‐pyrosequencing and high‐pressure liquid chromatography (HPLC) at five stations in summer 2010. Three stations (T1; T5; T7) were influenced by Polar Water, characterized by cold water with lower salinity (<33) and different extents of ice concentrations. Atlantic Water influenced the other two stations (T6; T9). While T6 was located in the mixed water zone characterized by cold water with intermediate salinity (~33) and high ice concentrations, T9 was located in warm water with high salinity (~35) and no ice‐coverage at all. General trends in community structure according to prevailing environmental settings, observed with both methods, coincided well. At two stations, T1 and T7, characterized by lower ice concentrations, diatoms (Fragilariopsis sp., Porosira sp., Thalassiosira spp.) dominated the protist community. The third station (T5) was ice‐covered, but has been ice‐free for ~4 weeks prior to sampling. At this station, dinoflagellates (Dinophyceae 1, Woloszynskia sp. and Gyrodinium sp.) were dominant, reflecting a post‐bloom situation. At station T6 and T9, the protist communities consisted mainly of picoeukaryotes, e.g., Micromonas spp. Based on our results, 454‐pyrosequencing has proven to be an adequate tool to provide comprehensive information on the composition of protist communities. Furthermore, this study suggests that a snap‐shot of a few, but well‐chosen samples can provide an overview of community structure patterns and succession in a dynamic marine environment.     

Cadmium‐induced and trans‐generational changes in the cultivable and total seed endophytic community of Arabidopsis thaliana

Trans‐generational adaptation is important to respond rapidly to environmental challenges and increase overall plant fitness. Besides well‐known mechanisms such as epigenetic modifications, vertically transmitted endophytic bacteria might contribute to this process. The cultivable and total endophytic communities of several generations of Arabidopsis thaliana seeds harvested from plants exposed to cadmium (Cd) or not exposed were investigated. The diversity and richness of the seed endophytic community decreased with an increasing number of generations. Aeromicrobium and Pseudonocardia were identified as indicator species in seeds from Cd‐exposed plants, while Rhizobium was abundantly present in both seed types. Remarkably, Rhizobium was the only genus that was consistently detected in seeds of all generations, which suggests that the phenotypic characteristics were more important as selection criteria for which bacteria are transferred to the next plant generation than the actual genera. Production of IAA was an important trait for endophytes from both seed types, while ACC deaminase activity and Cd tolerance were mainly associated with seed endophytes from Cd‐exposed plants. Understanding how different factors influence the seed endophytic community can help us to improve seed quality and plant growth through different biotechnological applications.     

Amplicon‐pyrosequencing‐based detection of compositional shifts in bryophyte‐associated fungal communities along an elevation gradient

Although bryophytes are a dominant vegetation component of boreal and alpine ecosystems, little is known about their associated fungal communities. HPLC assays of ergosterol (fungal biomass) and amplicon pyrosequencing of the ITS2 region of rDNA were used to investigate how the fungal communities associated with four bryophyte species changed across an elevational gradient transitioning from conifer forest to the low‐alpine. Fungal biomass and OTU richness associated with the four moss hosts did not vary significantly across the gradient (P > 0.05), and both were more strongly affected by host and tissue type. Despite largely constant levels of fungal biomass, distinct shifts in community composition of fungi associated with Hylocomium, Pleurozium and Polytrichum occurred between the elevation zones of the gradient. This likely is a result of influence on fungal communities by major environmental factors such as temperature, directly or indirectly mediated by, or interacting with, the response of other components of the vegetation (i.e. the dominant trees). Fungal communities associated with Dicranum were an exception, exhibiting spatial autocorrelation between plots, and no significant structuring by elevation. Nevertheless, the detection of distinct fungal assemblages associated with a single host growing in different elevation zones along an elevational gradient is of particular relevance in the light of the ongoing changes in vegetation patterns in boreal and alpine systems due to global climate warming.     

Molecular and morphological diversity of Trebouxia microalgae in sphaerothallioid Circinaria spp. lichens1

Three vagrant (Circinaria hispida, Circinaria gyrosa, and Circinaria sp. ‘paramerae’) and one crustose (semi‐vagrant, Circinaria sp. ‘oromediterranea’) lichens growing in very continental areas in the Iberian Peninsula were selected to study the phycobiont diversity. Mycobiont identification was checked using nrITS DNA barcoding: Circinaria sp. ‘oromediterranea’ and Circinaria sp. ‘paramerae’ formed a new clade. Phycobiont diversity was analyzed in 50 thalli of Circinaria spp. using nrITS DNA and LSU rDNA, with microalgae coexistence being found in all the species analyzed by Sanger sequencing. The survey of phycobiont diversity showed up to four different Trebouxia spp. as the primary phycobiont in 20 thalli of C. hispida, in comparison with the remaining Circinaria spp., where only one Trebouxia was the primary microalga. In lichen species showing coexistence, some complementary approaches are needed (454 pyrosequencing and/or ultrastructural analyses). Five specimens were selected for high‐throughput screening (HTS) analyses: 22 Trebouxia OTUs were detected, 10 of them not previously known. TEM analyses showed three different cell morphotypes (Trebouxia sp. OTU A12, OTU S51, and T. cretacea) whose ultrastructure is described here in detail for the first time. HTS revealed a different microalgae pool in each species studied, and we cannot assume a specific pattern between these pools and the ecological and/or morphological characteristics. The mechanisms involved in the selection of the primary phycobiont and the other microalgae by the mycobiont are unknown, and require complex experimental designs. The systematics of the genus Circinaria is not yet well resolved, and more analyses are needed to establish a precise delimitation of the species.     

Plant host and soil origin influence fungal and bacterial assemblages in the roots of woody plants

Microbial communities in plant roots provide critical links between above‐ and belowground processes in terrestrial ecosystems. Variation in root communities has been attributed to plant host effects and microbial host preferences, as well as to factors pertaining to soil conditions, microbial biogeography and the presence of viable microbial propagules. To address hypotheses regarding the influence of plant host and soil biogeography on root fungal and bacterial communities, we designed a trap‐plant bioassay experiment. Replicate Populus, Quercus and Pinus plants were grown in three soils originating from alternate field sites. Fungal and bacterial community profiles in the root of each replicate were assessed through multiplex 454 amplicon sequencing of four loci (i.e., 16S, SSU, ITS, LSU rDNA). Soil origin had a larger effect on fungal community composition than did host species, but the opposite was true for bacterial communities. Populus hosted the highest diversity of rhizospheric fungi and bacteria. Root communities on Quercus and Pinus were more similar to each other than to Populus. Overall, fungal root symbionts appear to be more constrained by dispersal and biogeography than by host availability.     

A metagenomics‐based approach to the top‐down effect on the detritivore food web: a salamanders influence on fungal communities within a deciduous forest

The flow of energy within an ecosystem can be considered either top‐down, where predators influence consumers, or bottom‐up, where producers influence consumers. Plethodon cinereus (Red‐backed Salamander) is a terrestrial keystone predator who feeds on invertebrates within the ecosystem. We investigated the impact of the removal of P. cinereus on the detritivore food web in an upland deciduous forest in northwest Ohio, U.S.A. A total of eight aluminum enclosures, each containing a single P. cinereus under a small log, were constructed in the deciduous forest. On Day 1 of the experiment, four salamanders were evicted from four of the eight enclosures. Organic matter and soil were collected from the center of each enclosure at Day 1 and Day 21. From each sample, DNA was extracted, fungal‐specific amplification performed, and 454 pyrosequencing was used to sequence the nuclear ribosomal internal transcribed spacer (ITS2) region and partial ribosomal large subunit (LSU). Changes in overall fungal community composition or species diversity were not statistically significant between treatments. Statistically significant shifts in the most abundant taxonomic groups of fungi were documented in presence but not absence enclosures. We concluded that P. cinereus does not affect the overall composition or diversity of fungal communities, but does have an impact on specific groups of fungi. This study used a metagenomics‐based approach to investigate a missing link among a keystone predator, P. cinereus, invertebrates, and fungal communities, all of which are critical in the detritivore food web.     

Relationships among wood‐boring beetles,fungi, and the decomposition of forest biomass

A prevailing paradigm in forest ecology is that wood‐boring beetles facilitate wood decay and carbon cycling, but empirical tests have yielded mixed results. We experimentally determined the effects of wood borers on fungal community assembly and wood decay within pine trunks in the southeastern United States. Pine trunks were made either beetle‐accessible or inaccessible. Fungal communities were compared using culturing and high‐throughput amplicon sequencing (HTAS) of DNA and RNA. Prior to beetle infestation, living pines had diverse fungal endophyte communities. Endophytes were displaced by beetle‐associated fungi in beetle‐accessible trees, whereas some endophytes persisted as saprotrophs in beetle‐excluded trees. Beetles increased fungal diversity several fold. Over forty taxa of Ascomycota were significantly associated with beetles, but beetles were not consistently associated with any known wood‐decaying fungi. Instead, increasing ambrosia beetle infestations caused reduced decay, consistent with previous in vitro experiments that showed beetle‐associated fungi reduce decay rates by competing with decay fungi. No effect of bark‐inhabiting beetles on decay was detected. Platypodines carried significantly more fungal taxa than scolytines. Molecular results were validated by synthetic and biological mock communities and were consistent across methodologies. RNA sequencing confirmed that beetle‐associated fungi were biologically active in the wood. Metabarcode sequencing of the LSU/28S marker recovered important fungal symbionts that were missed by ITS2, though community‐level effects were similar between markers. In contrast to the current paradigm, our results indicate ambrosia beetles introduce diverse fungal communities that do not extensively decay wood, but instead reduce decay rates by competing with wood decay fungi.     

Experimental warming alters the community composition,diversity, and N2 fixation activity of peat moss (Sphagnum fallax) microbiomes

Sphagnum‐dominated peatlands comprise a globally important pool of soil carbon (C) and are vulnerable to climate change. While peat mosses of the genus Sphagnum are known to harbor diverse microbial communities that mediate C and nitrogen (N) cycling in peatlands, the effects of climate change on Sphagnum microbiome composition and functioning are largely unknown. We investigated the impacts of experimental whole‐ecosystem warming on the Sphagnum moss microbiome, focusing on N₂ fixing microorganisms (diazotrophs). To characterize the microbiome response to warming, we performed next‐generation sequencing of small subunit (SSU) rRNA and nitrogenase (nifH) gene amplicons and quantified rates of N₂ fixation activity in Sphagnum fallax individuals sampled from experimental enclosures over 2 years in a northern Minnesota, USA bog. The taxonomic diversity of overall microbial communities and diazotroph communities, as well as N₂ fixation rates, decreased with warming (p < 0.05). Following warming, diazotrophs shifted from a mixed community of Nostocales (Cyanobacteria) and Rhizobiales (Alphaproteobacteria) to predominance of Nostocales. Microbiome community composition differed between years, with some diazotroph populations persisting while others declined in relative abundance in warmed plots in the second year. Our results demonstrate that warming substantially alters the community composition, diversity, and N₂ fixation activity of peat moss microbiomes, which may ultimately impact host fitness, ecosystem productivity, and C storage potential in peatlands.     

Mitochondrial DNA and Y‐chromosome diversity in East Adriatic sheep

Variation in mitochondrial DNA (mtDNA) and Y‐chromosome haplotypes was analysed in nine domestic sheep breeds (159 rams) and 21 mouflon ( Ovis musimon) sampled in the East Adriatic. Mitochondrial DNA analyses revealed a high frequency of type B haplotypes, predominantly in European breeds, and a very low frequency of type A haplotypes, which are more frequent in some Asian breeds. Mitochondrial haplotype Hmt‐3 was the most frequent (26.4%), and 37.1%, 20.8% and 7.6% of rams had haplotypes one, two and three mutations remote from Hmt‐3 respectively. In contrast, Y‐chromosome analyses revealed extraordinary paternal allelic richness: HY‐6, 89.3%; HY‐8, 5.0%; HY‐18, 3.1%; HY‐7, 1.3%; and HY‐5, 1.3%. In fact, the number of haplotypes observed is comparable to the number found in Turkish breeds and greater than the number found in European breeds so far. Haplotype HY‐18 (A‐oY1/135‐SRYM18), identified here for the first time, provides a link between the haplotype HY‐12 (A‐oY1/139‐SRYM18) found in a few rams in Turkey and haplotype HY‐9 (A‐oY1/131‐SRYM18) found in one ram in Ethiopia. All mouflons had type B mtDNA haplotypes, including the private haplotype (Hmt‐55), and all were paternally monomorphic for haplotype HY‐6. Our data support a quite homogeneous maternal origin of East Adriatic sheep, which is a characteristic of European breeds. At the same time, the high number of haplotypes found was surprising and intriguing, and it begs for further analysis. Simultaneous analysis of mtDNA and Y‐chromosome information allowed us to detect a large discrepancy between maternal and paternal lineages in some populations. This is most likely the result of breeder efforts to ‘upgrade’ local populations using rams with different paternal origins.     

The hindgut lumen prokaryotic microbiota of the termite Reticulitermes flavipes and its responses to dietary lignocellulose composition

Reticulitermes flavipes (Isoptera: Rhinotermitidae) is a highly eusocial insect that thrives on recalcitrant lignocellulosic diets through nutritional symbioses with gut‐dwelling prokaryotes and eukaryotes. In the R. flavipes hindgut, there are up to 12 eukaryotic protozoan symbionts; the number of prokaryotic symbionts has been estimated in the hundreds. Despite its biological relevance, this diverse community, to date, has been investigated only by culture‐ and cloning‐dependent methods. Moreover, it is unclear how termite gut microbiomes respond to diet changes and what roles they play in lignocellulose digestion. This study utilized high‐throughput 454 pyrosequencing of 16S V5‐V6 amplicons to sample the hindgut lumen prokaryotic microbiota of R. flavipes and to examine compositional changes in response to lignin‐rich and lignin‐poor cellulose diets after a 7‐day feeding period. Of the ~475 000 high‐quality reads that were obtained, 99.9% were annotated as bacteria and 0.11% as archaea. Major bacterial phyla included Spirochaetes (24.9%), Elusimicrobia (19.8%), Firmicutes (17.8%), Bacteroidetes (14.1%), Proteobacteria (11.4%), Fibrobacteres (5.8%), Verrucomicrobia (2.0%), Actinobacteria (1.4%) and Tenericutes (1.3%). The R. flavipes hindgut lumen prokaryotic microbiota was found to contain over 4761 species‐level phylotypes. However, diet‐dependent shifts were not statistically significant or uniform across colonies, suggesting significant environmental and/or host genetic impacts on colony‐level microbiome composition. These results provide insights into termite gut microbiome diversity and suggest that (i) the prokaryotic gut microbiota is much more complex than previously estimated, and (ii) environment, founding reproductive pair effects and/or host genetics influence microbiome composition.     

孑遗植物桫椤内生真菌的多样性与群落组成

桫椤为木本蕨类,在世界范围内属于濒危的孑遗植物,关于桫椤内生真菌的研究还未见报道。本文基于形态学特征和DNA序列分析技术,探讨了桫椤内生真菌多样性和群落组成。研究发现,桫椤组织中蕴藏着丰富的真菌资源,内生真菌定殖率达到87.6%;从桫椤的234个生活组织块中,分离到内生真菌291株,被鉴定为64个分类单元;桫椤组织中常见真菌种类大约占内生真菌的60%,其中铺散内曼菌Nemania diffusa表现出明显的分布优势;内生真菌总体多样性指数达到3.53的较高水平,并且表现出叶轴>根部>羽片>叶柄>茎皮的分布规律;分析不同组织中内生真菌的相似性系数和聚类结果,发现桫椤的组织性质影响内生真菌的组成和分布;根据物种累积曲线,现有的研究样本中超过80%的内生真菌已经被分离到,研究结果能够反映桫椤内生真菌的多样性和群落组成。通过分析桫椤的叶柄凋落物中真菌定殖率、丰度、多样性、相似性和真菌种类等特征性数值,表明凋落的桫椤组织中真菌的分布特征已经发生了明显改变。     

Effects of temperature and medium composition on inhibitory activities of gossypol‐related compounds against aflatoxigenic fungi

Aims

To investigate the effects of temperature and medium composition on growth/aflatoxin inhibitory activities of terpenoids gossypol, gossypolone and apogossypolone against Aspergillus flavus and A. parasiticus.

Methods and Results

The compounds were tested at a concentration of 100 μg ml^?1 in a Czapek Dox (Czapek) agar medium at 25, 31 and 37°C. Increased incubation temperature marginally increased growth inhibition caused by these compounds, but reduced the aflatoxin inhibition effected by gossypol. Gossypolone and apogossypolone retained good aflatoxin inhibitory activity against A. flavus and A. parasiticus at higher incubation temperatures. However, increased temperature also significantly reduced aflatoxin production in control cultures. The effects of the terpenoids on fungal growth and aflatoxin production against the same fungi were also determined in Czapek, Czapek with a protein/amino acid addendum and yeast extract sucrose (YES) media. Growth of these fungi in the protein‐supplemented Czapek medium or in the YES medium greatly reduced the growth inhibition effects of the terpenoids. Apogossypolone displayed strong anti‐aflatoxigenic activity in the Czapek medium, but this activity was significantly reduced in the protein‐amended Czapek and YES media. Gossypol, which displayed little to no aflatoxin inhibitory activity in the Czapek medium, did yield significant anti‐aflatoxigenic activity in the YES medium.

Conclusions

Incubation temperature and media composition are important parameters involved in the regulation of aflatoxin production in A. flavus and A. parasiticus. These parameters also affect the potency of growth and aflatoxin inhibitory activities of these gossypol‐related compounds against aflatoxigenic fungi.

Significance and Impact of the Study

Studies utilizing gossypol‐related compounds as inhibitory agents of biological activities should be interpreted with caution due to compound interaction with multiple components of the test system, especially serum proteins.     

Total lipid and fatty acid composition of seaweeds for the selection of species for oil‐based biofuel and bioproducts

We investigated the potential of seaweeds as feedstock for oil‐based products, and our results support macroalgae (seaweeds) as a biomass source for oil‐based bioproducts including biodiesel. Not only do several seaweeds have high total lipid content above 10% dry weight, but in the brown alga Spatoglossum macrodontum 50% of these lipids are in the form of extractable fatty acids. S. macrodontum had the highest fatty acid content (57.40 mg g^?1 dw) and a fatty acid profile rich in saturated fatty acids with a high content of C18:1, which is suitable as a biofuel feedstock. Similarly, the green seaweed Derbesia tenuissima has high levels of fatty acids (39.58 mg g^?1 dw), however, with a high proportion of PUFA (n‐3) (31% of total lipid) which are suitable as nutraceuticals or fish oil replacements. Across all species of algae the critical parameter of fatty acid content (measured as fatty acid methyl esters, FAME) was positively correlated (R² = 0.67) with total lipid content. However, the proportion of fatty acids to total lipid decreased markedly with total lipid content, generally between 30% and 50%, making it an inaccurate measure of the potential to identify seaweeds suitable for oil‐based bioproducts. Finally, we quantified within species variation of fatty acids across locations and sampling periods supporting either environmental effects on quantitative fatty acid profiles, or genotypes with specific quantitative fatty acid profiles, thereby opening the possibility to optimize the fatty acid content and quality for oil production through specific culture conditions and selective breeding.     

Stream invertebrate diversity reduces with invasion of river banks by non‐native plants

1. Invasion of riparian zones by non‐native plants is a global issue and commonly perceived as a challenge for river and fishery managers, but the type and extent of ecological changes induced by such invasions remain poorly understood. Established effects on sediment delivery, allochthonous inputs, and channel shading could potentially alter aquatic macroinvertebrate assemblages, with implications for in‐stream ecological quality.
2. We assessed responses in the diversity, quality, and heterogeneity of stream macroinvertebrate communities to riparian invasion by non‐native plants. Macroinvertebrates were collected from 24 sites on low order streams in central and southern Scotland during spring and autumn. The effect of invasive non‐native plants (INNP) on macroinvertebrates was assessed relative to that of local physical and chemical factors.
3. Invasive non‐native plants cover was associated with stronger effects than other factors on local diversity of macroinvertebrates (33% reduction at the highest INNP cover) but also increased macroinvertebrate abundance across sites. Invaded sites were also associated with lower macroinvertebrate biomonitoring scores. Community composition differed between invaded and uninvaded sites in autumn, but not in spring. However, INNP influence on macroinvertebrate composition was generally secondary to that of physicochemical variables (e.g. channel shade, substrate diversity).
4. We demonstrate that the influence of INNP extends beyond well‐known impacts on plant communities to reductions mainly in stream macroinvertebrate diversity. Combined with the negative impact on pollution‐sensitive macroinvertebrate taxa this raises concerns over the ecological health of streams with heavily invaded riparian zones. Our findings suggest that efforts to improve low order streams by actively managing severe riparian invasions are merited, but the size and uncertainty of the likely ecological gains must also be evaluated against the effort involved.