Habitat partitioning in native Geranium species through reproductive interference.

BACKGROUND AND AIMS: Heterospecific pollen transfer may reduce the fitness of recipient species, a phenomenon known as reproductive interference. A theoretical study has predicted that distributions of species pairs affected by reproductive interference may be syntopic under negligible reproductive interference, sympatric but with partitioning at small spatial scale (i.e. allotopic) under weak interference, or exclusive when reproductive interference is strong. Verifying these predictions is essential for evaluation of the applicability of reproductive interference as a general assembly rule of biological communities. The aim of this study was to test these predictions in two sympatrically distributed wild Geranium species, G. thunbergii and G. wilfordii. METHODS: To measure the effect of reproductive interference, the associations between the relative abundance of the counterpart species and seed set in the focal species, and seed set reduction following mixed pollination, were analysed. The possibility of hybridization with viable offspring was examined by genotyping plants in the field and after mixed pollination. Fertility of putative hybrids was based on their seed set and the proportion of pollen grains with apertural protrusions. A transect study was conducted to examine spatial partitioning, and possible influences of environmental conditions (canopy openness and soil moisture content) on partitioning between the species were analysed. KEY RESULTS: Neither abundance of the counterpart species nor heterospecific pollen deposition significantly affected seed set in the focal species, and hybridization between species was almost symmetrical. Putative hybrids had low fertility. The two species were exclusively distributed at small scale, although environmental conditions were not significantly different between them. CONCLUSIONS: The allotopy of the two species may be maintained by relatively weak reproductive interference through bidirectional hybridization. Re-evaluation of hybridization may allow ongoing or past reproductive interference to be recognized and provide insight into the distributional relationships between the interacting plants.

Inhomogeneity of sediment samples in analysis of hexabromocyclododecane.

The repeatability test of the analytical method for hexabromocyclododecane (HBCD) was conducted with sediment sample. The maximum HBCD concentration exceeded the minimum by a factor of 90 even though the identical sediment samples were used. Therefore, we examined which step of the analytical method was the factor causing variability. We examined the blank test, and confirmation test of the extraction and purified procedure. From these results, we confirmed that there was nothing wrong with the accuracy of our analytical method. These results indicate that the variability of HBCD concentration in the repeatability test was attributed not to the analytical method, but to the inhomogeneity of the sediment sample. Aluminum, silicon, and organic carbon in sediments were measured to compare the variability of these concentrations with that of HBCD concentration. These concentrations were similar values within identical samples which showed variability in HBCD concentration. HBCD concentration in several samples did not correlate with organic carbon content. These results suggests that sediment samples were homogeneous by itself, and HBCD was heterogeneously distributed in spite of homogeneity of organic carbon in sediment. The sediment sample with variability in HBCD concentration showed different HBCD diastereomer compositions in identical sediment. It implies that the sample contained HBCD derived from different histories or point sources. Even though we increased sample amounts to analyze the homogeneity of the sample, HBCD concentration varied within identical samples if the sample had a hot spot. Past monitoring data may contain overestimation or underestimation of HBCD concentration in sediment.

Skewed male reproductive success and pollen transfer in a small fragmented population of the heterodichogamous tree Machilus thunbergii.

Heterodichogamy is defined as the presence of two flower morphs that exhibit the male and female functions at different times among individuals within a population. Heterodichogamy is regarded as an adaptation to promote outcrossing through enhanced inter-morph mating, together with a 1:1 morph ratio. However, in highly fragmented populations, the morph ratio may be more likely to be biased by stochastic events. In such a situation, individuals of a minority morph within a population are expected to have higher reproductive success than those of a majority morph, which may suffer from pollen shortages of the minority morph. In this paper, we evaluated mating patterns and male reproductive success in a highly fragmented population of Machilus thunbergii, a putative heterodichogamous evergreen laurel tree. Results of paternity analysis indicated that the selfing rate was not clearly different between the two morphs. In contrast, the proportion of intra-morph mating was higher in the majority-morph (MM) mother trees than in the minority-morph (MF) mother trees. Bayesian estimated male reproductive success indicated that male reproductive success was higher in minority-morph (MF) than in majority-morph (MM) mother trees. These findings indicate that (1) the majority morph mothers, suffering a shortage of the opposite morph pollen, could partly compensate for the reduced reproductive success by intra-morph mating rather than by selfing, and (2) negative-frequency dependent selection may be involved in the maintenance of the two morphs.

A Bayesian approach for estimating hexabromocyclododecane (HBCD) diastereomer compositions in water using data below limit of quantification.

We developed a statistical model that quantitatively incorporated the stochastic fluctuations of values, which were estimated as Bayesian credible intervals (BCIs), to analyze environmental monitoring data. We used α-hexabromocyclododecane (α-HBCD) diastereomer compositions of water samples that included data points below limit of quantification. To avoid replacing "not detected (ND)" values with irrelevant values in data analysis, we substituted ND with observed values from the measurement system upon examination of the model. In our study, it was assumed that the magnitude of stochastic fluctuations of observed values in environmental samples was identical to that in iterative measurements of a standard solution at the lowest concentration. Using this model, α-HBCD diastereomer compositions could be estimated along with BCIs even for samples collected from sites where concentrations of α-HBCD were ND or near limit of quantification. The brackish areas in our study showed relatively wide ranges in composition for the 95% BCIs compared with samples from fresh water areas. In the brackish areas, concentrations of HBCD were frequently ND or near limit of quantification. Using this model, it was unnecessary to replace ND with zero or limit of quantification in data analysis, and an environmental assessment could be achieved using all of the data. Therefore, this model is considered to be a widely applicable approach in the analysis of environmental monitoring data including ND.

Spatial Distribution of Flower Color Induced by Interspecific Sexual Interaction.

Understanding the mechanisms shaping the spatiotemporal distribution of species has long been a central concern of ecology and evolutionary biology. Contemporary patterns of plant assemblies suggest that sexual interactions among species, i.e., reproductive interference, lead to the exclusive distributions of closely related species that share pollinators. However, the fitness consequences and the initial ecological/evolutionary responses to reproductive interference remain unclear in nature, since reproductive isolation or allopatric distribution has already been achieved in the natural community. In Japan, three species of blue-eyed grasses (Sisyrinchium) with incomplete reproductive isolation have recently colonized and occur sympatrically. Two of them are monomorphic with white flowers, whereas the other exhibits heritable color polymorphism (white and purple morphs). Here we investigated the effects of the presence of two monomorphic species on the distribution and reproductive success of color morphs. The frequency and reproductive success of white morphs decreased in area where monomorphic species were abundant, while those of purple morphs did not. The rate of hybridization between species was higher in white morphs than in the purple ones. Resource competition and habitat preference seemed not to contribute to the spatial distribution and reproductive success of two morphs. Our results supported that color-dependent reproductive interference determines the distribution of flower color polymorphism in a habitat, implying ecological sorting promoted by pollinator-mediated reproductive interference. Our study helps us to understand the evolution and spatial structure of flower color in a community.

Flower color polymorphism maintained by overdominant selection in Sisyrinchium sp.

Negative frequency-dependent selection derived from positive frequency-dependent foraging is the best-known selection force maintaining genetic polymorphism within a population. However, in flowering plants, positive frequency-dependent foraging by pollinators is expected to accelerate the loss of low-frequency morphs by conferring a fitness advantage to the common morph, leading to monomorphism. In Japan, a non-native species, Sisyrinchium sp., exhibits conspicuous flower color polymorphism within a population comprising both purple morphs (homozygous recessive) and white morphs (heterozygous or homozygous dominant). Here we quantified genotype-specific reproductive success in order to reveal the contribution of overdominant selection on the maintenance of flower color polymorphism in this species. In artificial pollination experiments using individuals with identified genotypes, female reproductive success was higher in the heterozygote than in either homozygote. The frequency of purple morphs in natural populations (ca. 31%) is similar to the frequency predicted by overdominant selection (25%). Our results suggest that overdominant selection contributes to the maintenance of color morphs in the natural population of this species.

Unionid Freshwater Mussels in Irrigation Ditches are Affected by Physical Environmental Factors and Proximity to Paddy Fields.

Irrigation ditches are the major habitat of lotic unionid mussels in Japan. To conserve and rebuild irrigation ditches facilitating mussel conservation, suitable physical environments must be clarified. The effect on mussels of paddy fields connected to ditches also needs to be determined. In this study, physical environmental factors that affect the density of unionid mussels were studied in irrigation ditches in Hikone City, Shiga Prefecture, Japan, to examine whether mussel densities were higher around paddy fields. Generalized linear models were used to analyze physical and paddy field environmental variables affecting mussel density. Our results show that sediment type, sediment softness, water depth, and flow velocity of irrigation ditches affect the density of unionid mussels; the effects of each environment factor and their relative importance differed by species. Specifically, the density of Nodularia douglasiae biwae was higher in ditches with sand-gravel sediment, soft sediment, and not adjoining paddy fields. The density of Pronodularia japanensis was higher in ditches with sand-gravel sediment and not adjoining paddy fields. The density of Lanceolaria grayana was higher in ditches with high flow velocity, not adjoining paddy fields, close to Lake Biwa. The density of Sinanodonta japonica was higher in ditches with mud sediment, shallow depth, high flow velocity, and not adjoining paddy fields. The densities of all four species were lower in irrigation ditches that were closely connected to paddy fields, suggesting that paddy drainage water negatively affects the survival of the mussels.

Sexual shape dimorphism accelerated by male-male competition, but not prevented by sex-indiscriminate parental care in dung beetles (Scarabaeidae).

Dimorphic sexual differences in shape and body size are called sexual dimorphism and sexual size dimorphism, respectively. The degrees of both dimorphisms are considered to increase with sexual selection, represented by male-male competition. However, the degrees of the two dimorphisms often differ within a species. In some dung beetles, typical sexual shape dimorphisms are seen in male horns and other exaggerated traits, although sexual size dimorphism looks rare. We hypothesized that the evolution of this sexual shape dimorphism without sexual size dimorphism is caused by male-male competition and their crucial and sex-indiscriminate provisioning behaviors, in which parents provide the equivalent size of brood ball with each of both sons and daughters indiscriminately. As a result of individual-based model simulations, we show that parents evolve to provide each of sons and daughters with the optimal amount of resource for a son when parents do not distinguish the sex of offspring and males compete for mates. This result explains why crucial and sex-indiscriminate parental provisioning does not prevent the evolution of sexual shape dimorphism. The model result was supported by empirical data of Scarabaeidae beetles. In some dung beetles, sexual size dimorphism is absent, compared with significant sexual size dimorphism in other horned beetles, although both groups exhibit similar degrees of sexual shape dimorphism in male horns and other exaggerated traits.

Associations between co-detected respiratory viruses in children with acute respiratory infections.

Viruses are the major etiological agents of acute respiratory infections (ARIs) in young children. Although respiratory virus co-detections are common, analysis of combinations of co-detected viruses has never been conducted in Japan. Nineteen respiratory viruses or subtypes were surveyed using multiplex real-time PCR on 1,044 pediatric (patient age < 6 years) ARI specimens collected in Osaka City, Japan between January 2010 and December 2011. In total, 891 specimens (85.3%) were virus positive (1,414 viruses were detected), and 388 of the virus-positive specimens (43.5%, 388/891) were positive for multiple viruses. The ratio of multiple/total respiratory virus-positive specimens was high in children aged 0-35 months. Statistical analyses revealed that human bocavirus 1 and human adenovirus were synchronously co-detected. On the other hand, co-detections of human parainfluenza virus type 1 (HPIV-1) with HPIV-3, HPIV-3 with human metapneumovirus (hMPV), hMPV with respiratory syncytial virus A (RSV A), hMPV with influenza virus A (H1N1) 2009 (FLUA (H1N1) 2009), RSV A with RSV B, and human rhinovirus and FLUA (H1N1) 2009 were exclusive. These results suggest that young children (<3 years) are highly susceptible to respiratory viruses, and some combinations of viruses are synchronously or exclusively co-detected.

Distribution and pollutant load of hexabromocyclododecane (HBCD) in sewage treatment plants and water from Japanese Rivers.

Hexabromocyclododecane diastereoisomers (α-, ß-, γ-, δ-, and ε-HBCD) were investigated in river water, sewage influent, and sewage effluent from the Yodo River basin, Japan. The mean annual values of HBCDs ranged from 0.19 to 14ngL(-1) in river water. We observed that the concentrations of HBCDs in the brackish water area were low compared with that in the fresh water area. It was implied that, when the flow of the river stagnated in the estuarine area, HBCDs settled with suspended matter because of their hydrophobic character. In the sewage treatment plants, HBCDs ranged from 16 to 400ngL(-1) in sewage influent, whereas they ranged from 0.39 to 12ngL(-1) in sewage effluent. Over 90% of HBCDs were removed from the wastewater in the sewage treatment plants. By using these results, we estimated the pollutant load of HBCDs that flows into Osaka Bay from the study area. It was estimated that approximately 15kg of HBCDs flow into Osaka Bay from the study area in a year. This value is five orders of magnitude lower than the 2010 market demand for HBCDs (3019 metrictons) in Japan.

Distribution of Capnocytophaga canimorsus in dogs and cats with genetic characterization of isolates.

Capnocytophaga canimorsus, which is often found in the oral cavities of dogs and cats, is sometimes transmitted to humans, causing severe infection. To elucidate the risk of C. canimorsus in humans and animals, this study was undertaken to characterize this bacterium epidemiologically and genetically. We examined the distribution of C. canimorsus in dogs and cats, and analyzed the correlation between the presence of bacteria and individual factors statistically. We also compared C. canimorsus isolates genetically using 16S rRNA gene sequence analysis and pulsed-field gel electrophoresis (PFGE). C. canimorsus was detected in 76 of 109 dogs (69.7%) and 57 of 104 cats (54.8%). A relation between C. canimorsus presence and some individual factors was detected both in dogs and cats, but the predictive factors of carrying the bacterium differed between dogs and cats. 16S rRNA gene sequences from C. canimorsus isolates in this study were combined with previously published sequences to assess their intra-specific phylogeny. Results show that C. canimorsus is classifiable into two main groups (I and II) with differing γ-glutamyl aminopeptidase activity. Strains from human patients belonged unevenly to group I, possibility suggesting that group I can be transmitted to humans and group II is indigenous only to the oral cavities of dogs and cats. PFGE genotyping showed high discriminatory power, and the dendrogram accorded with genetic segregation between isolates of group I and II. Sma I-digest PFGE developed for this study is useful as a molecular typing method for additional epidemiological and phylogenetic studies of C. canimorsus.

Frequent respiratory viral infections in a young child in a 27-month follow-up study.

INTRODUCTION: Viruses are major aetiological agents of acute respiratory infection in young children. Although many studies have reported detection and analysis of respiratory viruses in sporadic cases, there have been few follow-up studies of individuals. The purpose of this study was to investigate the frequency of respiratory viral infections in a young child and to examine the duration of viral genome detection in clinical specimens. CASE PRESENTATION: A total of 284 nasal swabs were collected during symptomatic (196 specimens) and asymptomatic (88 specimens) periods of respiratory symptoms from a young female child (from 4 months to 31 months of age, who was admitted to a nursery school at 9 months). Multiplex real-time PCR for 19 respiratory viruses or subtypes was performed. One hundred and ninety-eight of the tested specimens were virus positive (69.7 %) (symptomatic periods, 149/196, 76.0 %; asymptomatic periods, 49/88, 55.7 %). Rhinovirus was the most frequently detected (26 times). Long durations of detection were observed for human coronavirus NL63 (30 days), rhinovirus (28 days) and human bocavirus 1 (22 days). CONCLUSION: Young children living in a group context have a high risk of respiratory virus infections, especially rhinovirus. In some instances, viral genomes were detectable for about 1 month by PCR.

Biofilm formation and resistance to benzalkonium chloride in Listeria monocytogenes isolated from a fish processing plant.

Listeria monocytogenes is a foodborne pathogen that causes the potentially life-threatening illness listeriosis. Previously, a few clones of L. monocytogenes persisting in a cold-smoked fish processing plant were isolated from the plant's products continuously. To evaluate the role of biofilms in the persistence of L. monocytogenes strains specific to this plant, the abilities of the persistent strain (PS) and transient strain (TS) of L. monocytogenes found in this plant to form biofilms were compared, as was resistance to the sanitizing effects of benzalkonium chloride (BC). The PS produced more biofilm than the TS in 48 h. The half-maximal effective concentration (EC50), the BC concentration at which the ATP bioluminescence of each bacterial strain decreased by 50 % relative to its maximum activity, was about 150-fold higher in the PS than in the TS. In contrast, when these values were measured in organisms in a planktonic state, the EC50 of the PS was only 2.2-fold higher than that of the TS. Extracellular polymeric substances (EPS) were extracted from biofilms, and the glucose content of these biofilms was determined with the phenol-sulfuric acid method to estimate the quantity of EPS. The total amount of EPS in the PS biofilm was higher than that in the TS biofilm. These findings suggest that the PS produces greater amounts of biofilm and EPS than the TS, which results in greater resistance of the PS to disinfectants. The persistence of the strain in the fish processing plant might be attributable to these properties.

Occurrence of fluoroquinolones and fluoroquinolone-resistance genes in the aquatic environment.

Fluoroquinolones (FQs) have been detected in aquatic environments in several countries. Long-term exposure to low levels of antimicrobial agents provides selective pressure, which might alter the sensitivity of bacteria to antimicrobial agents in the environment. Here, we examined FQ levels and the resistance of Escherichia coli (E. coli) to FQs by phenotyping and genotyping. In the aquatic environment in Osaka, Japan, ciprofloxacin, enoxacin, enfloxacin, lomefloxacin, norfloxacin, and ofloxacin were detected in concentrations ranging from 0.1 to 570 ng L(-1). FQ-resistant E. coli were also found. Although no obvious correlation was detected between the concentration of FQs and the presence of FQ-resistant E. coli, FQ-resistant E. coli were detected in samples along with FQs, particularly ciprofloxacin and ofloxacin. Most FQ-resistant E. coli carried mutations in gyrA, parC, and parE in quinolone resistance-determining regions. No mutations in gyrB were detected in any isolates. Amino acid changes in these isolates were quite similar to those in clinical isolates. Six strains carried the plasmid-mediated quinolone resistance determinant qnrS1 and expressed low susceptibility to ciprofloxacin and nalidixic acid: the minimum inhibitory concentrations ranged from 0.25 µg mL(-1) for ciprofloxacin, and from 8 to 16 µg mL(-1) for nalidixic acid. This finding confirmed that plasmids containing qnr genes themselves did not confer full resistance to quinolones. Because plasmids are responsible for much of the horizontal gene transfer, these genes may transfer and spread in the environment. To our knowledge, this is the first report of plasmid-mediated quinolone resistance determinant qnrS1 in the aquatic environment, and this investigation provides baseline data on antimicrobial resistance profiles in the Osaka area.

Safer DNA extraction from plant tissues using sucrose buffer and glass fiber filter.

For some plant species, DNA extraction and downstream experiments are inhibited by various chemicals such as polysaccharides and polyphenols. This short communication proposed an organic-solvent free (except for ethanol) extraction method. This method consists of an initial washing step with STE buffer (0.25 M sucrose, 0.03 M Tris, 0.05 M EDTA), followed by DNA extraction using a piece of glass fiber filter. The advantages of this method are its safety and low cost. The purity of the DNA solution obtained using this method is not necessarily as high as that obtained using the STE/CTAB method, but it is sufficient for PCR experiments. These points were demonstrated empirically with two species, Japanese speedwell and common dandelion, for which DNA has proven difficult to amplify via PCR in past studies.

Increase of GII.2 norovirus infections during the 2009-2010 season in Osaka City, Japan.

During the 2009-2010 season, a significant numerical increase of genotype GII.2 norovirus (NoV)-associated outbreaks was observed in Osaka City, Japan. The most common genotype in that season was GII.2 (44.6%), followed by GII.4 (39.2%). Mostly, GII.2 strains were associated with outbreaks in children and with person-to-person contact. The National Infectious Disease Surveillance Center reported that GII.2 NoV infections were widespread in Japan in that season. Comparative phylogenetic analysis of RNA-dependent RNA polymerase (RdRp) and capsid sequences revealed that this GII.2 epidemic resulted from two genetic strains. The first, GII.2p2 strains, had an identical genotype in the RdRp and capsid genes. GII.2p2 strains in the 2009-2010 season were a different genetic cluster from the strains of spring 2004, the previous epidemic of GII.2 NoV, but showed no unique amino acid change. The second, GII.2 chimera virus (GII.2p16), had GII.16 RdRp and GII.2 capsid genotypes, suggesting prior recombination at the junction of ORF1 and ORF2. GII.2p16 strains had four significant amino acid changes in the P2 subdomain, suggesting antigenic changes. Before the 2009-2010 season, GII.2 chimera viruses had been observed only sporadically. This spreading of GII.2p16 strains in the 2009-2010 season might be the first epidemic of GII.2 chimera virus. This study revealed that the NoV epidemic in the 2009-2010 season differed considerably from the prior season, when GII.4 was predominant. Furthermore, GII.2 strains persisted in human populations by drastic recombination and gradual accumulation of mutations, indicating a prevalent pattern of non-GII.4 genotypes with genetic evolution.

Effective range of reproductive interference exerted by an alien dandelion, Taraxacum officinale, on a native congener.

Reproductive interference (RI), defined as the fitness cost of interspecific sexual interactions, such as interspecific pollen transfer (IPT) in plants, is ecologically important. Theoretically, RI could result in competitive exclusion, as it operates in a frequency-dependent manner. Additionally, IPT may have a greater range than resource competition, although information about the range of IPT is lacking. In the present study, we measured the range of IPT exerted by Taraxacum officinale (an alien species) on a native dandelion, T. japonicum. We used two approaches. In one, we analyzed the RI effect on a native seed set at three spatial scales. In the second, we tracked IPT from alien to native flower heads using fluorescent pigments as markers. We estimated that pollination distances were in the order of several meters. These distances exceeded the mean distance from each native plant to the nearest alien. As hypothesized, the effect of RI reached farther than neighboring individuals. These data indicate the spatial range from which alien dandelions should be removed to allow the conservation of natives.

Detection and quantitative analysis of human bocavirus associated with respiratory tract infection in Osaka City, Japan.

HBoV was initially identified in patients with RTI in 2005. Since its discovery, there have been continual reports concerning HBoV detection and its prevalence. In this study of clinical specimens from young children, real-time PCR was undertaken to examine whether HBoV infection is associated with RTI and to support quantitative analysis of HBoV in these patients. In all, 376 specimens were collected from patients with RTI during April 2006-October 2008. Analyses revealed HBoV in 59 specimens (15.7%). Of HBoV-positive patients, children under the age of 3 years comprised 94.9%. Of the HBoV-positive samples, 47.5% were codetected with other respiratory viruses (dual infection, 27; triple infection, 1). During the study period, the numbers and rate of detection of HBoV were high mainly around May. Statistical analyses showed that the detection rate of HBoV during April-June was higher than during other months. Moreover, the viral load was greater in subjects with infection with HBoV alone than in subjects with mixed respiratory viral infections. Considering these results together, HBoV is probably associated with RTI in young children. However, the pathogenesis of this infection and the importance of the high rate of co-infection remain uncertain. Additional epidemiologic information and further analyses are necessary to clarify the virological characteristics and the linkage of HBoV to disease.

Reconsiderations on evaluating methodology of repellent effects: validation of indices and statistical analyses.

Many pest repellents are in household use, and a variety of procedures, indices, and statistical tests have been used to examine their effectiveness. However, the effect of action mechanism on these indices has not been adequately discussed. In addition, the interreplication variance of data have not been taken into account in some cases. This study considered the validity of tests used to evaluate repellents and proposed methods to statistically evaluate them. First, various repellents with different mechanisms of action were virtually assumed, and their repelling processes were formulated as differential equations. Numerical and analytical analyses of the formulae revealed that, in some action mechanisms, the indices could be altered by parameters other than the repellent effect and that the indices should be used only for repellents of a specific mechanism. Furthermore, the effect of an actual repellent, by using two different experimental designs and the maize weevil, Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae), were evaluated. This demonstrated that the evaluated effectiveness could vary with experimental design and suggested that the design should reflect the situation in which repellents are actually used. Therefore, statistical methods to quantify the effectiveness of repellents are proposed herein and applied to the results of the above-mentioned experiments. Particular emphasis was placed on the importance of incorporating the interreplication variance in statistical models and estimating confidence intervals. Finally, the validity and limitations of evaluation using indices of repellents' effects, and the identification of important criteria that should be considered when assessing the effects of repellents also are discussed.