Quantification of Alternaria,Cladosporium, Fusarium and Penicillium verrucosum in Conventional and Organic Grains by qPCR

We analysed the levels of Alternaria, Cladosporium, Fusarium and Penicillium verrucosum in grain samples harvested in 2011 and 2012 from conventional and organic farms using qPCR. In general, both Alternaria and Cladosporium occurred in all cereal grains in the highest quantities, followed by P. verrucosum and Fusarium. Alternaria, Cladosporium and P. verrucosum had the highest levels in crop mixtures, barley and rye and lower levels in wheat, while Fusarium levels were the highest in crop mixtures and wheat. The levels of Alternaria and P. verrucosum were higher in organic rye and wheat than conventional grains. Although the level of Fusarium was higher in conventional than organic rye, opposite results were obtained for crop mixtures. A positive correlation was found between Alternaria, Cladosporium and P. verrucosum, indicating that similar factors might affect the distribution of these fungi in grains.     

Annual plants change in size over a century of observations

Studies have documented changes in animal body sizes over the last century, but very little is known about changes in plant sizes, even though reduced plant productivity is potentially responsible for declines in size of other organisms. Here, I ask whether warming trends in the Great Basin have affected plant size by measuring specimens preserved on herbarium sheets collected between 1893 and 2011. I asked how maximum and minimum temperatures, precipitation, and the Pacific Decadal Oscillation (PDO) in the year of collection affected plant height, leaf size, and flower number, and asked whether changes in climate resulted in decreasing sizes for seven annual forbs. Species had contrasting responses to climate factors, and would not necessarily be expected to respond in parallel to climatic shifts. There were generally positive relationships between plant size and increased minimum and maximum temperatures, which would have been predicted to lead to small increases in plant sizes over the observation period. While one species increased in size and flower number over the observation period, five of the seven species decreased in plant height, four of these decreased in leaf size, and one species also decreased in flower production. One species showed no change. The mechanisms behind these size changes are unknown, and the limited data available on these species (germination timing, area of occupancy, relative abundance) did not explain why some species shrank while others grew or did not change in size over time. These results show that multiple annual forbs are decreasing in size, but that even within the same functional group, species may have contrasting responses to similar environmental stimuli. Changes in plant size could have cascading effects on other members of these communities, and differential responses to directional change may change the composition of plant communities over time.     

Depolarization‐induced suppression of a glycinergic synapse in the superior olivary complex by endocannabinoids

The neuronal endocannabinoid system is known to depress synaptic inputs retrogradely in an activity‐dependent manner. This mechanism has been generally described for excitatory glutamatergic and inhibitory GABAergic synapses. Here, we report that neurones in the auditory brainstem of the Mongolian gerbil (Meriones unguiculatus) retrogradely regulate the strength of their inputs via the endocannabinoid system. By means of whole‐cell patch‐clamp recordings, we found that retrograde endocannabinoid signalling attenuates both glycinergic and glutamatergic post‐synaptic currents in the same types of neurones. Accordingly, we detected the cannabinoid receptor 1 in excitatory and inhibitory pre‐synapses as well as the endocannabinoid‐synthesising enzymes (diacylglycerol lipase α/β, DAGLα/β) post‐synaptically through immunohistochemical stainings. Our study was performed with animals aged 10–15 days, that is, in the time window around the onset of hearing. Therefore, we suggest that retrograde endocannabinoid signalling has a role in adapting inputs during the functionally important switch from spontaneously generated to sound‐related signals.

     

Expression of TaCYP78A3, a gene encoding cytochrome P450 CYP78A3 protein in wheat (Triticum aestivum L.), affects seed size

Several studies have described quantitative trait loci (QTL) for seed size in wheat, but the relevant genes and molecular mechanisms remain largely unknown. Here we report the functional characterization of the wheat TaCYP78A3 gene and its effect on seed size. TaCYP78A3 encoded wheat cytochrome P450 CYP78A3, and was specifically expressed in wheat reproductive organs. TaCYP78A3 activity was positively correlated with the final seed size. Its silencing caused a reduction of cell number in the seed coat, resulting in an 11% decrease in wheat seed size, whereas TaCYP78A3 over‐expression induced production of more cells in the seed coat, leading to an 11–48% increase in Arabidopsis seed size. In addition, the cell number in the final seed coat was determined by the TaCYP78A3 expression level, which affected the extent of integument cell proliferation in the developing ovule and seed. Unfortunately, TaCYP78A3 over‐expression in Arabidopsis caused a reduced seed set due to an ovule developmental defect. Moreover, TaCYP78A3 over‐expression affected embryo development by promoting embryo integument cell proliferation during seed development, which also ultimately affected the final seed size in Arabidopsis. In summary, our results indicated that TaCYP78A3 plays critical roles in influencing seed size by affecting the extent of integument cell proliferation. The present study provides direct evidence that TaCYP78A3 affects seed size in wheat, and contributes to an understanding of the cellular basis of the gene influencing seed development.     

Phylogenetics of tribe Sabiceeae (Ixoroideae,Rubiaceae) revisited,with a new subgeneric classification for Sabicea

Tribe Sabiceeae (Ixoroideae, Rubiaceae) has undergone recent taxonomical changes with the incorporation of the related genera Ecpoma, Pseudosabicea and Stipularia into the type genus Sabicea. We use phylogenetic analysis and morphological data to verify the relationships among members of the tribe, including the most comprehensive taxon sampling of the tribe to date with 74 of 145 species. Sequence data from the nuclear internal transcribed spacer (ITS) and three plastid markers (petD, rps16, trnT–F) were used to infer relationships among the members of the tribe. Individual analyses using maximum likelihood, parsimony and Bayesian approaches reveal several supported clades: the former genus Stipularia is resolved as a monophyletic unit, but Ecpoma is monophyletic only if Sabicea urbaniana and Sabicea xanthotricha are included (corresponding to Sabicea subgenus Stipulariopsis sensu Wernham). Pseudosabicea is biphyletic, with one clade corresponding to section Anisophyllae of Hallé (1964) and the other one to the other sections (Floribundae and Sphaericae) of the genus. Eleven morphological characteristics were recorded for all species studied and seven have been mapped onto the phylogenetic tree to study their evolution in the group and assess their value for the classification of Sabicea s.l. Finally, our study shows that a combination of diagnostic characteristics should be used to differentiate each group and we propose to recognise four subgenera in Sabicea.     

A combined morphological,ultrastructural, molecular,and biochemical study of the peculiar family Gomontiellaceae (Oscillatoriales) reveals a new cylindrospermopsin‐producing clade of cyanobacteria

Members of the morphologically unusual cyanobacterial family Gomontiellaceae were studied using a polyphasic approach. Cultured strains of Hormoscilla pringsheimii, Starria zimbabweënsis, Crinalium magnum, and Crinalium epipsammum were thoroughly examined, and the type specimen of the family, Gomontiella subtubulosa, was investigated. The results of morphological observations using both light microscopy and transmission electron microscopy were consistent with previous reports and provided evidence for the unique morphological and ultrastructural traits of this family. Analysis of the 16S rRNA gene confirmed the monophyletic origin of non‐marine repre‐sentatives of genera traditionally classified into this family. The family was phylogenetically placed among other groups of filamentous cyanobacterial taxa. The presence of cellulose in the cell wall was analyzed and confirmed in all cultured Gomontiellaceae members using Fourier transform infrared spectroscopy and fluorescence microscopy. Evaluation of toxins produced by the studied strains revealed the hepatotoxin cylindrospermopsin (CYN) in available strains of the genus Hormoscilla. Production of this compound in both Hormoscilla strains was detected using high‐performance liquid chromatography in tandem with high resolution mass spectrometry and confirmed by positive PCR amplification of the cyrJ gene from the CYN biosynthetic cluster. To our knowledge, this is the first report of CYN production by soil cyanobacteria, establishing a previously unreported CYN‐producing lineage. This study indicates that cyanobacteria of the family Gomontiellaceae form a separate but coherent cluster defined by numerous intriguing morphological, ultrastructural, and biochemical features, and exhibiting a toxic potential worthy of further investigation.     

Geographic mosaic of symbiont selectivity in a genus of epiphytic cyanolichens

In symbiotic systems, patterns of symbiont diversity and selectivity are crucial for the understanding of fundamental ecological processes such as dispersal and establishment. The lichen genus Nephroma (Peltigerales, Ascomycota) has a nearly cosmopolitan distribution and is thus an attractive model for the study of symbiotic interactions over a wide range of spatial scales. In this study, we analyze the genetic diversity of Nephroma mycobionts and their associated Nostoc photobionts within a global framework. The study is based on Internal Transcribed Spacer (ITS) sequences of fungal symbionts and tRNA^Leu (UAA) intron sequences of cyanobacterial symbionts. The full data set includes 271 Nephroma and 358 Nostoc sequences, with over 150 sequence pairs known to originate from the same lichen thalli. Our results show that all bipartite Nephroma species associate with one group of Nostoc different from Nostoc typically found in tripartite Nephroma species. This conserved association appears to have been inherited from the common ancestor of all extant species. While specific associations between some symbiont genotypes can be observed over vast distances, both symbionts tend to show genetic differentiation over wide geographic scales. Most bipartite Nephroma species share their Nostoc symbionts with one or more other fungal taxa, and no fungal species associates solely with a single Nostoc genotype, supporting the concept of functional lichen guilds. Symbiont selectivity patterns within these lichens are best described as a geographic mosaic, with higher selectivity locally than globally. This may reflect specific habitat preferences of particular symbiont combinations, but also the influence of founder effects.     

Validation of a cheap and simple nondestructive method for obtaining AFLPs and DNA sequences (mitochondrial and nuclear) in amphibians

The use of nondestructive methods for obtaining DNA from amphibians (e.g. buccal swabs) allows genetic studies to be performed without affecting the survival of the studied individuals. In this study, we compared two methods of nondestructive DNA sampling, buccal swabs and interdigital membrane or toe‐clipping, in several amphibian species of different size: Rhinella spinulosa, R. atacamensis, six species of the genus Telmatobius and Pleurodema thaul. We evaluated the integrity of the DNA extracted by sequencing fragments of mitochondrial and nuclear genes and by generating amplified fragment length polymorphisms markers (AFLPs). In all cases, we obtained an adequate amount of DNA (mean range 55–298 ng/μL). We obtained identical DNA sequences from buccal swab and interdigital membrane/toe‐clip for all individuals. The differences in the coding of AFLP markers between the tissues were similar to those reported for replicas of the same type of sample in similar analyses in other species of amphibians. In conclusion, the use of buccal swabs is a trustworthy and inexpensive method to obtain DNA for mitochondrial and nuclear sequencing and AFLP analyses. Given the types of markers evaluated, buccal swabs may be used for phylogenetic, phylogeographic and population genetic studies, even in small amphibians (<33 mm).     

Species discrimination in Sisyrinchium (Iridaceae): assessment of DNA barcodes in a taxonomically challenging genus

DNA barcoding aims to develop an efficient tool for species identification based on short and standardized DNA sequences. In this study, the DNA barcode paradigm was tested among the genera of the tribe Sisyrinchieae (Iridoideae). Sisyrinchium, with more than 77% of the species richness in the tribe, is a taxonomically complex genus. A total of 185 samples belonging to 98 species of Sisyrinchium, Olsynium, Orthrosanthus and Solenomelus were tested using matK, trnH‐psbA and internal transcribed spacer (ITS). Candidate DNA barcodes were analysed either as single markers or in combination. Detection of a barcoding gap, similarity‐based methods and tree‐based analyses were used to assess the discrimination efficiency of DNA barcodes. The levels of species identification obtained from plastid barcodes were low and ranged from 17.35% to 20.41% for matK and 5.11% to 7.14% for trnH‐psbA. The ITS provided better results with 30.61–38.78% of species identified. The analyses of the combined data sets did not result in a significant improvement in the discrimination rate. Among the tree‐based methods, the best taxonomic resolution was obtained with Bayesian inference, particularly when the three data sets were combined. The study illustrates the difficulties for DNA barcoding to identify species in evolutionary complex lineages. Plastid markers are not recommended for barcoding Sisyrinchium due to the low discrimination power observed. ITS gave better results and may be used as a starting point for species identification.     

Cranial morphology and taxonomic resolution of some dolphin taxa (Delphinidae) in Australian waters,with a focus on the genus Tursiops

Phylogenetic relationships in the family Delphinidae have been widely debated. We examined 347 skulls of Tursiops, Stenella, Delphinus, Steno, Lagenodelphis, and Sousa in order to resolve the phylogenetic position of Australian species of Tursiops. Five Tursiops type specimens were included. Cranial morphology was described using 2‐dimensional (2‐D) and 3‐dimensional geometric morphometrics (3‐GM), counts and categorical data. Analyses showed a clear morphological separation of Tursiops, including type specimens, from other genera. The three Stenella species did not cluster together. Stenella attenuata clustered with Delphinus delphis, and Stenella coeruleoalba with Lagenodelphis hosei. Length and width of the skull and rostrum were important discriminators in both methods. For 3‐D data, round vs. angular posterior skull shape distinguished some genera. Taxa that overlapped in the multivariate analyses had different mean tooth counts. Our study challenges genetic studies that identified Tursiops as polyphyletic, with T. aduncus closer to S. attenuata.     

Phylogenetic Position of Parasitic Chytrids on Diatoms: Characterization of a Novel Clade in Chytridiomycota

Chytrids are true fungi that reproduce with posteriorly uniflagellate zoospores. In the last decade, environmental DNA surveys revealed a large number of uncultured chytrids as well as undescribed order‐level novel clades in Chytridiomycota. Although many species have been morphologically described, only some DNA sequence data of parasitic chytrids are available from the database. We herein discuss five cultures of parasitic chytrids on diatoms Aulacoseira spp. and Asterionella formosa. In order to identify the chytrids examined, thallus morphologies were observed using light microscopy. We also conducted a phylogenetic analysis using 18S, 5.8S, and 28S rDNA sequences to obtain their phylogenetic positions. Based on their morphological characteristics, two cultures parasitic on As. formosa were identified as Rhizophydium planktonicum and Zygorhizidium planktonicum. The other three cultures infecting Aulacoseira spp. (two on Aulacoseira ambigua and the other on Aulacoseira granulata) were regarded as Zygorhizidium aff. melosirae. The results of the molecular phylogenetic analysis revealed that R. planktonicum belonged to the known order Chytridiales, while the two species of Zygorhizidium were placed in a novel clade that was previously reported as an undescribed clade composed of only the environmental sequences of uncultured chytrids.     

Amplification of ABA biosynthesis and signaling through a positive feedback mechanism in seeds

Abscisic acid is an essential hormone for seed dormancy. Our previous study using the plant gene switch system, a chemically induced gene expression system, demonstrated that induction of 9‐cis‐epoxycarotenoid dioxygenase (NCED), a rate‐limiting ABA biosynthesis gene, was sufficient to suppress germination in imbibed Arabidopsis seeds. Here, we report development of an efficient experimental system that causes amplification of NCED expression during seed maturation. The system was created with a Triticum aestivum promoter containing ABA responsive elements (ABREs) and a Sorghum bicolor NCED to cause ABA‐stimulated ABA biosynthesis and signaling, through a positive feedback mechanism. The chimeric gene pABRE:NCED enhanced NCED and ABF (ABRE‐binding factor) expression in Arabidopsis Columbia‐0 seeds, which caused 9‐ to 73‐fold increases in ABA levels. The pABRE:NCED seeds exhibited unusually deep dormancy which lasted for more than 3 months. Interestingly, the amplified ABA pathways also caused enhanced expression of Arabidopsis NCED5, revealing the presence of positive feedback in the native system. These results demonstrated the robustness of positive feedback mechanisms and the significance of NCED expression, or single metabolic change, during seed maturation. The pABRE:NCED system provides an excellent experimental system producing dormant and non‐dormant seeds of the same maternal origin, which differ only in zygotic ABA. The pABRE:NCED seeds contain a GFP marker which enables seed sorting between transgenic and null segregants and are ideal for comparative analysis. In addition to its utility in basic research, the system can also be applied to prevention of pre‐harvest sprouting during crop production, and therefore contributes to translational biology.     

Extreme protomeric development in a burlingiid trilobite from Cambrian glacial erratics of Denmark

Pengia Geyer & Corbacho is a Cambrian burlingiid trilobite with fused trunk segments devoid of any articulation in the anamorphic and epimorphic phases of development. The type species is Pengia fusilis (Peng et al.) from the Wanshania wanshanensis Zone of China. Here we describe a second species, Pengia palsgaardia sp. nov., from the Lejopyge laevigata Zone of the Paradoxides forchhammeri Superzone. It comes from a glacial erratic in Denmark which probably originated in the Alum Shale Formation of Västergötland, Sweden. Pengia palsgaardia is a large burlingiid (~10 mm in length), with 14 fused segments in the trunk whose boundaries are marked by ridges. The axis is narrow, with the axial furrows faintly indicated or effaced across the median. Laterally along the axis and the tapering glabella, symmetrical globular lobes are developed that are pinched at their base. During ontogeny the glabellar furrows are pit‐like adaxially but shallow towards the axial furrow as the globular lobes develop. Their pit‐like appearance in Pengia palsgaardia and some other burlingiid species is not considered similar to the condition seen in oryctocephalid trilobites. A median preglabellar ridge resembling that of Schmalenseeia Moberg develops late in ontogeny but in early ontogeny the preglabellar field resembles that of Burlingia Walcott, Alumenella Geyer & Corbacho and Niordilobites Geyer & Corbacho. This gives Pengia a more basal position in the schmalenseeid lineage, outside the derived Schmalenseeia. In mature specimens the facial sutures in P. palsgaardia are fused, but an ocular suture may have been present. During ontogeny Pengia would have gone through the anamorphic and protomeric protaspid segmental conditions, but articulation between either the cephalon and pygidium, or pygidium and thoracic segments of the trunk never developed so it did not progress beyond the protaspid phase. This extreme protomeric development is considered to be a derived feature in Pengia.     

Population genetic data of a model symbiotic cnidarian system reveal remarkable symbiotic specificity and vectored introductions across ocean basins

The Aiptasia–Symbiodinium symbiosis is a promising model for experimental studies of cnidarian–dinoflagellate associations, yet relatively little is known regarding the genetic diversity of either symbiotic partner. To address this, we collected Aiptasia from 16 localities throughout the world and examined the genetic diversity of both anemones and their endosymbionts. Based on newly developed SCAR markers, Aiptasia consisted of two genetically distinct populations: one Aiptasia lineage from Florida and a second network of Aiptasia genotypes found at other localities. These populations did not conform to the distributions of described Aiptasia species, suggesting that taxonomic re‐evaluation is needed in the light of molecular genetics. Associations with Symbiodinium further demonstrated the distinctions among Aiptasia populations. According to 18S RFLP, ITS2‐DGGE and microsatellite flanker region sequencing, Florida anemones engaged in diverse symbioses predominantly with members of Symbiodinium Clades A and B, but also C, whereas anemones from elsewhere harboured only S. minutum within Clade B. Symbiodinium minutum apparently does not form a stable symbiosis with other hosts, which implies a highly specific symbiosis. Fine‐scale differences among S. minutum populations were quantified using six microsatellite loci. Populations of S. minutum had low genotypic diversity and high clonality (R = 0.14). Furthermore, minimal population structure was observed among regions and ocean basins, due to allele and genotype sharing. The lack of genetic structure and low genotypic diversity suggest recent vectoring of Aiptasia and S. minutum across localities. This first ever molecular‐genetic study of a globally distributed cnidarian and its Symbiodinium assemblages reveals host–symbiont specificity and widely distributed populations in an important model system.