Biology subjects

Ushio, M.

Publications and source records attributed to Ushio, M..

29 records · Page 2Linked to original sources

bioRxiv2021

Quantitative assessment of multiple fish species around artificial reefs using environmental DNA metabarcoding

Since the early 1970s, many artificial reefs (ARs) have been deployed in Japanese coastal waters to create fisheries grounds. Recently, researchers began to use environmental DNA (eDNA) methods for biodiversity monitoring of aquatic species. A metabarcoding approach using internal standard DNAs (i.e., quantitative MiSeq sequencing) makes it possible to monitor eDNA concentrations of multiple species simultaneously. This method can improve the efficiency of monitoring AR effects on fishes. Our study investigated distributions of marine fishes at ARs and surrounding stations in the open oceanographic environment of Tateyama Bay, central Japan, using quantitative MiSeq sequencing and echo sounder survey. Using the quantitative metabarcoding method, we found higher quantities of fish eDNAs at the ARs than at surrounding stations and different fish species compositions between them. Comparisons with eco sounder survey also showed positive correlations between fish eDNA concentration and echo intensity, which indicates a highly localized signal of eDNA at each sampling station. These results suggest that quantitative MiSeq sequencing is a promising technique to complement conventional methods to monitor distributions of multiple fish species.

ecology↗

bioRxiv2020

Evaluation of seasonal dynamics of fungal DNA assemblages in a flow-regulated stream in a restored forest using eDNA metabarcoding

Investigation of seasonal variation in fungal communities is essential for understanding biodiversity and ecosystem functions. However, the conventional sampling method, with substrate removal and high spatial heterogeneity of community composition, makes surveying the seasonality of fungal communities challenging. Recently, water environmental DNA (eDNA) analysis has been explored for its utility in biodiversity surveys. In this study, we assessed whether the seasonality of fungal communities can be detected by monitoring eDNA in a forest stream. We conducted monthly water sampling in a forest stream over two years and used DNA metabarcoding to identify fungal eDNA. The stream water contained DNA from functionally diverse aquatic and terrestrial fungi, such as plant decomposers, parasites, and mutualists. The variation in the fungal assemblage showed a regular annual periodicity, meaning that the assemblages in a given season were similar, irrespective of the year or sampling. Furthermore, the strength of the annual periodicity varied among functional groups. Our results suggest that forest streams may act as a "trap" for terrestrial fungal DNA derived from different habitats, allowing the analysis of fungal DNA in stream water to provide information about the temporal variation in fungal communities in both the aquatic and the surrounding terrestrial ecosystems.

ecology↗

bioRxiv2020

Interaction capacity underpins community diversity

How patterns in community diversity emerge is a long-standing question in ecology. Theories and experimental studies suggested that community diversity and interspecific interactions are interdependent. However, evidence from multitaxonomic, high-diversity ecological communities is lacking because of practical challenges in characterizing speciose communities and their interactions. Here, I analyzed time-varying causal interaction networks that were reconstructed using 1197 species, DNA-based ecological time series taken from experimental rice plots and empirical dynamic modeling, and show that species interaction capacity, namely, the sum of interaction strength that a single species gives and receives, underpins community diversity. As community diversity increases, the number of interactions increases exponentially but the mean species interaction capacity of a community becomes saturated, weakening interaction among species. These patterns are explicitly modeled with simple mathematical equations, based on which I propose the "interaction capacity hypothesis," namely, that species interaction capacity and network connectance are proximate drivers of community diversity. Furthermore, I show that total DNA copy number and temperature influence species interaction capacity and connectance nonlinearly, explaining a large proportion of diversity patterns observed in various systems. The interaction capacity hypothesis enables mechanistic explanations of community diversity, and how species interaction capacity is determined is a key question in ecology.

ecology↗

bioRxiv2019

Environmental DNA analysis shows high potential as a tool for estimating intraspecific genetic diversity in a wild fish population

Environmental DNA (eDNA) analysis has recently been used as a new tool for estimating intraspecific diversity. However, whether known haplotypes contained in a sample can be detected correctly using eDNA-based methods has been examined only by an aquarium experiment. Here, we tested whether the haplotypes of Ayu fish (Plecoglossus altivelis altivelis) detected in a capture survey could also be detected from an eDNA sample derived from the field that contained various haplotypes with low concentrations and foreign substances. A water sample and Ayu specimens collected from a river on the same day were analysed by eDNA analysis and Sanger sequencing, respectively. The 10 L water sample was divided into 20 filters for each of which 15 PCR replications were performed. After high-throughput sequencing, denoising was performed using two of the most widely used denoising packages, UNOISE3 and DADA2. Of the 42 haplotypes obtained from the Sanger sequencing of 96 specimens, 38 (UNOISE3) and 41 (DADA2) haplotypes were detected by eDNA analysis. When DADA2 was used, except for one haplotype, haplotypes owned by at least two specimens were detected from all the filter replications. This study showed that the eDNA analysis for evaluating intraspecific genetic diversity provides comparable results for large-scale capture-based conventional methods, suggesting that it could become a more efficient survey method for investigating intraspecific genetic diversity in the field.

molecular biology↗

bioRxiv2019

Idea Paper: Predicting culturability of microbes from population dynamics under field conditions

Isolation and cultivation of microbes from environmental samples have been fundamental and important for species identification and investigating functions and ecology of target microbes. While cultivation and isolation of microbes are not easy, the natural environment can \"culture\" any endemic microbes, and thus key information for culturing and isolating microbes may be encoded in the natural population dynamics of microbes. In this paper, I present the idea that culturability of microbes may be inferred by quantifying dynamics properties of microbes using nonlinear time series analytical tools. To briefly demonstrate the idea, I analyzed high-frequency, quantitative microbial time series obtained for artificial rice plots established at Kyoto University, Japan. I selected bacterial phyla that included sufficient numbers of microbial taxa, and analyzed 398 microbial taxa using empirical dynamic modeling. The nine phyla analyzed generally followed a similar pattern: many microbial taxa fell into the \"Simple\" dynamics category, and a small proportion of taxa were categorized in \"Simple but nonlinear\" or \"Nearly random\" dynamics categories. The present analysis suggested that many microbes in the study system might be cultivated by modifying a relatively small number of conditions. However, the present idea as well as the result is preliminary and premature, and more precise taxonomic information (i.e., species-level identification) and a culturability dataset will help to validate the idea. If the present idea was found to be valid, a priori evaluation of the culturability of microbes would become possible, which would avoid unnecessary costs (labor, time and money) of attempts to cultivate microbes.

ecology↗

bioRxiv2019

Dynamic and synergistic influences of air temperature and rainfall on general flowering in a Bornean lowland tropical forest

Supra-annually synchronized flowering events occurring in tropical forests in Southeast Asia, known as general flowering (GF), are \"spectacular and mysterious\" forest events. Recently, studies that combined novel molecular techniques and model-based theoretical approaches suggested that cool temperature and drought synergistically drove GF. Although the novel approaches advanced our understanding of GF, it is usually difficult to know whether the mathematical formulation reasonably well represents the complex and dynamic processes involved in GF. In the present study, we collected a 17-year set of community-wide phenology data during 1993 to 2011 from Lambir Hills National Park in Borneo, Malaysia, and analyzed it using a model-free approach, empirical dynamic modeling (EDM), that does not rely on specific assumptions about the underlying mechanisms, to overcome and complement the previous limitations. By using EDM, we found that GF in the forest in Lambir Hills National Park is synergistically driven by cool air temperature and drought, which is consistent with the previous studies. Also, we found that cumulative meteorological variables, rather than instantaneous values, drive GF with delayed effects, which is also consistent with the previous studies. Interestingly, the present study showed that effects of cumulative meteorological variables on GF changed through time, which implies that the relationship between GF and meteorological variables may be influenced by other factors such as plant/soil nutrient resource dynamics. Future studies integrating novel mathematical/statistical frameworks, long-term and large spatial scale ecosystem monitoring and molecular phenology data are promising for better understanding and fore-casting of GF events in tropical forests in Southeast Asia.

ecology↗

bioRxiv2018

Celestially determined annual seasonality of equatorial tropical rain forests

O_LIIntra-annual periodicity of canopy photosynthetic activity and leaf development has been documented in seasonal and weakly-seasonal tropical forests in the Amazon and elsewhere. However, vegetative periodicity such as leaf flush and fall in apparently "aseasonal" equatorial tropical forests has not been well documented. Moreover, causal drivers of the vegetative periodicity in those forests have not been identified largely because of the difficulty in performing manipulative experiments targeting whole forest ecosystem dynamics. C_LIO_LIHere we show a distinct annual seasonality in canopy dynamics using a Fourier analysis with a statistical significance test on the long-term, fortnightly monitored dataset of leaf litterfall in nine evergreen tropical rain forests on Mount Kinabalu, Borneo. Statistically significant annual periodicity occurs across altitudes and soil types in all years irrespective of the year-to-year climatic variability, suggesting that fluctuations in regional climate rather than local micro-climatic, edaphic and/or biotic conditions cause the precise 1-year periodicity. C_LIO_LIWe examine climatic factors that have causative effects on the distinct 1-year periodicity using the spectrum convergent cross mapping that we developed in the present study to distinguish causal relationships from seasonality-driven synchronization. According to the analysis, we find that mean daily air temperature is most strongly, causatively related to the 1-year periodicity of leaf litterfall. However, knowledge on ecophysiolocial and molecular mechanisms underlying temperature-control of tropical tree growth is limited and further studies are required to understand the detailed mechanisms. C_LIO_LI(Synthesis) We suggest that intra-annual temperature changes in association with the movement of the intertropical convergence zone cause the distinct annual vegetative periodicity. Because vegetative periodicity can be transmitted to the dynamics of higher trophic levels through a trophic cascade, interactions between vegetative periodicity and daily air temperature, not rainfall, would more strongly cause changes in the dynamics of equatorial tropical rain forests. Our results show that clear vegetative periodicity (i.e., annual seasonality) can be found in equatorial tropical rain forests under diverse local environments, and that air temperature is a more important factor than the other climate variables in the climate-forest ecosystem interactions. C_LI

ecology↗

bioRxiv2018

Use of a filter cartridge combined with bead beating improves detection of microbial DNA from water samples

Microbial communities play an important role in driving the dynamics of aquatic ecosys-tems. As difficulties in DNA sequencing faced by microbial ecologists are continuously being reduced, sample collection methods and the choice of DNA extraction protocols are becoming more critical to the outcome of any sequencing study. In the present study, I added a manual bead-beating step in the protocol using a DNeasy(R) Blood & Tissue kit for DNA extraction from a filter cartridge (Sterivex filter cartridge) without breaking the cartridge unit (\"Beads\" method), and compared its performance with those of two other protocols that used the filter cartridge (\"NoBeads\" method, which was similar to the Beads method but without the bead-beating step, and \"PowerSoil\" method, which followed the manual of the PowerSoil DNA extraction kit after breaking apart the filter cartridge). Water samples were collected from lake and river ecosystems in Japan, and DNA was extracted using the three protocols. Then, the V4 region of prokaryotic 16S rRNA genes was amplified. In addition, internal standard DNAs were included in the DNA library preparation process to estimate the number of 16S rRNA gene copies. The DNA library was sequenced using Illumina MiSeq, and sequences were analyzed using the amplicon sequence variant (ASV) approach (DADA2). I found that, 1) the total DNA yields were highest with the Beads method, 2) the number of ASVs (a proxy for species richness) was also highest with the Beads method, 3) overall community compositions were significantly different among the three methods, and 4) the number of method-specific ASVs was highest with the Beads method. In conclusion, the inclusion of a bead-beating step performed inside the filter cartridge increased the DNA yield as well as the number of prokaryotic ASVs detected compared with the other two methods. Performing the bead-beating step inside the filter cartridge causes no dramatic increase in either handling time or processing cost. Therefore, this method has the potential to become one of the major choices when one aims to extract aquatic microbial DNAs.

ecology↗

bioRxiv2018

Evaluating intraspecific diversity of a fish population using environmental DNA: An approach to distinguish true haplotypes from erroneous sequences

Recent advances in environmental DNA (eDNA) analysis using high-throughput sequencing (HTS) provide a non-invasive way to evaluate the intraspecific genetic diversity of aquatic macroorganisms. However, erroneous sequences present in HTS data can result in false positive haplotypes; therefore, reliable strategies are necessary to eliminate such erroneous sequences when evaluating intraspecific genetic diversity using eDNA metabarcoding. In this study, we propose an approach combining denoising using amplicon sequence variant (ASV) method and the removal of haplotypes with low detection rates. A mixture of rearing water of Ayu (Plecoglossus altivelis altivelis) was used as an eDNA sample. In total, nine haplotypes of Ayu mitochondrial D-loop region were contained in the sample and amplified by two-step tailed PCR. The 15 PCR replicates indexed different tags were prepared from the eDNA sample to compare the detection rates between true haplotypes and false positive haplotypes. All PCR replications were sequenced by HTS, and the total number of detected true haplotypes and false positive haplotypes were compared with and without denoising using the two types of ASV methods, Divisive Amplicon Denoising Algorithm 2 (DADA2) and UNOISE3. The use of both ASV methods considerably reduced the number of false positive haplotypes. Moreover, all true haplotypes were detected in all 15 PCR, whereas false positive haplotypes had detection rates varying from 1/15 to 15/15. Thus, by removing haplotypes with lower detection rates than 15/15, the number of false positive haplotypes were further reduced. The approach proposed in this study successfully eliminated most of false positive haplotypes in the HTS data obtained from eDNA samples, which allowed us to improve the detection accuracy for evaluating intraspecific genetic diversity using eDNA analysis.

molecular biology↗

bioRxiv2017

Demonstration of the potential of environmental DNA as a tool for the detection of avian species

Birds play unique functional roles in the maintenance of ecosystems, such as pollina-tion and seed dispersal, and thus monitoring bird communities (e.g., monitoring bird species diversity) is a first step towards avoiding undesirable consequences of anthro-pogenic impacts on bird communities. In the present study, we hypothesized that birds, regardless of their main habitats, must have frequent contact with water and that tissues that contain their DNA that persists in the environment (environmental DNA; eDNA) could be used to detect the presence of avian species. To this end, we applied a set of universal PCR primers (MiBird, a modified version of fish/mammal universal primers) for metabarcoding avian eDNA. We confirmed the versatility of MiBird primers by performing in silico analyses and by amplifying DNAs extracted from bird tissues. Analyses of water samples from zoo cages of birds with known species composition suggested that the use of MiBird primers combined with the Il-lumina MiSeq platform could successfully detect avian species from water samples. Additionally, analysis of water samples collected from a natural pond detected five avian species common to the sampling areas. The present findings suggest that avian eDNA metabarcoding would be a complementary detection/identification tool in cases where visual detection and identification of bird species is difficult.

ecology↗

bioRxiv2017

Quantitative monitoring of multispecies fish environmental DNA using high-throughput sequencing

Effective ecosystem conservation and resource management require quantitative monitoring of biodiversity, including accurate descriptions of species composition and temporal variations of species abundance. Therefore, quantitative monitoring of biodiversity has been performed for many ecosystems, but it is often time- and effort-consuming and costly. Recent studies have shown that environmental DNA (eDNA), which is released to the environment from macro-organisms living in a habitat, contains information about species identity and abundance. Thus, analyzing eDNA would be a promising approach for more efficient biodiversity monitoring. In the present study, we added internal standard DNAs (i.e., known amounts of short DNA fragments from fish species that have never been observed in a sampling area) to eDNA samples, which were collected weekly from a coastal marine ecosystem in Maizuru-Bay, Kyoto, Japan (from April 2015 to March 2016), and performed metabarcoding analysis using Illumina MiSeq to simultaneously identify fish species and quantify fish eDNA copy numbers. A correction equation was obtained for each sample using the relationship between the number of sequence reads and the added amount of the standard DNAs, and this equation was used to estimate the copy numbers from the sequence reads of non-standard fish eDNA. The calculated copy numbers showed significant positive correlation with those determined by quantitative PCR, suggesting that eDNA metabarcoding with standard DNA enabled useful quantification of eDNA. Furthermore, for samples that show a high level of PCR inhibition, our method might allow more accurate quantification than qPCR because the correction equations generated using internal standard DNAs would include the effect of PCR inhibition. A single run of Illumina MiSeq produced > 70 quantitative fish eDNA time series in our study, showing that our method could contribute to more efficient and quantitative monitoring of biodiversity.

ecology↗