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.     

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.     

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).     

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.

     

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.     

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.     

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.     

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.     

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.     

Phylogeography of the freshwater raphidophyte Gonyostomum semen confirms a recent expansion in northern Europe by a single haplotype

Gonyostmum semen is a freshwater raphidophyte that has increased in occurrence and abundance in several countries in northern Europe since the 1980s. More recently, the species has expanded rapidly also in north‐eastern Europe, and it is frequently referred to as invasive. To better understand the species history, we have explored the phylogeography of G. semen using strains from northern Europe, United States, and Japan. Three regions of the ribosomal RNA gene (small subunit [SSU], internal transcribed spacer [ITS] and large subunit [LSU]) and one mitochondrial DNA marker (cox1) were analyzed. The SSU and partial LSU sequences were identical in all strains, confirming that they belong to the same species. The ITS region differentiated the American from the other strains, but showed high intra‐strain variability. In contrast, the mitochondrial marker cox1 showed distinct differences between the European, American, and Japanese strains. Interestingly, only one cox1 haplotype was detected in European strains. The overall low diversity and weak geographic structure within northern European strains supported the hypothesis of a recent invasion of new lakes by G. semen. Our data also show that the invasive northern European lineage is genetically distinct from the lineages from the other continents. Finally, we concluded that the mitochondrial cox1 was the most useful marker in determining large‐scale biogeographic patterns in this species.     

The Lactoperoxidase System: a Natural Biochemical Biocontrol Agent for Pre‐ and Postharvest Applications

Controlling pests in pre‐ and postharvest crops using natural and low‐impact products is a major challenge. The lactoperoxidase system is an enzymatic system that exists in all external secretions in mammals and is part of the non‐immune system. We tested its efficacy in in vitro microplates on Phytophthora infestans, Penicillium digitatum, Penicillium italicum, Penicillium expansum and Botrytis cinerea to determine the most suitable concentrations for use. Then, we verified its efficacy in planta under controlled conditions. Solutions prepared with 5.4 mm iodide and 1.2 mm thiocyanate and diluted threefold inhibited pathogen growth in vitro by 63–100%. Twofold‐diluted solutions protected potato plants against P. infestans by 60–74% under controlled conditions. Undiluted solution inhibited orange's and apple's postharvest pathogens in curative application with efficacy levels ranging between 84 and 95% in orange and between 63 and 74% in apple. 1.5‐fold concentrated solutions inhibited postharvest pathogens of apple in curative application with efficacy levels ranging between 84 and 92%. Our results also show that the oxidative stress response of fruit following wounding could interfere with ion efficiency. Our tests demonstrate for the first time that this biochemical method is as efficient as a conventional synthetic chemical method under controlled conditions.     

Clonal diversity driven by parasitism in a freshwater snail

One explanation for the widespread abundance of sexual reproduction is the advantage that genetically diverse sexual lineages have under strong pressure from virulent coevolving parasites. Such parasites are believed to track common asexual host genotypes, resulting in negative frequency‐dependent selection that counterbalances the population growth‐rate advantage of asexuals in comparison with sexuals. In the face of genetically diverse asexual lineages, this advantage of sexual reproduction might be eroded, and instead sexual populations would be replaced by diverse assemblages of clonal lineages. We investigated whether parasite‐mediated selection promotes clonal diversity in 22 natural populations of the freshwater snail Melanoides tuberculata. We found that infection prevalence explains the observed variation in the clonal diversity of M. tuberculata populations, whereas no such relationship was found between infection prevalence and male frequency. Clonal diversity and male frequency were independent of snail population density. Incorporating ecological factors such as presence/absence of fish, habitat geography and habitat type did not improve the predictive power of regression models. Approximately 11% of the clonal snail genotypes were shared among 2–4 populations, creating a web of 17 interconnected populations. Taken together, our study suggests that parasite‐mediated selection coupled with host dispersal ecology promotes clonal diversity. This, in return, may erode the advantage of sexual reproduction in M. tuberculata populations.     

Expression of a carotenoid‐modifying gene and evolution of red coloration in weaverbirds (Ploceidae)

Red carotenoid colours in birds are widely assumed to be sexually selected quality indicators, but this rests on a very incomplete understanding of genetic mechanisms and honesty‐mediating costs. Recent progress was made by the implication of the gene CYP2J19 as an avian carotenoid ketolase, catalysing the synthesis of red C4‐ketocarotenoids from yellow dietary precursors, and potentially a major mechanism behind red coloration in birds. Here, we investigate the role of CYP2J19 in the spectacular colour diversification of African weaverbirds (Ploceidae), represented by five genera and 16 species: eight red, seven yellow and one without carotenoid coloration. All species had a single copy of CYP2J19, unlike the duplication found in the zebra finch, with high expression in the retina, confirming its function in colouring red oil droplets. Expression was weak or undetected in skin and follicles of pigment‐depositing feather buds, as well as in beaks and tarsi, including those of the red‐billed quelea. In contrast, the hepatic (liver) expression of CYP2J19 was consistently higher (>14‐fold) in seven species with C4‐ketocarotenoid coloration than in species without (including one red species), an association strongly supported by a phylogenetic comparative analysis. The results suggest a critical role of the candidate ketolase, CYP2J19, in the evolution of red C4‐ketocarotenoid colour variation in ploceids. As ancestral state reconstruction suggests that ketocarotenoid coloration has evolved twice in this group (once in Euplectes and once in the Quelea/Foudia clade), we argue that while CYP2J19 has retained its ancestral role in the retina, it has likely been co‐opted for red coloration independently in the two lineages, via increased hepatic expression.