Double-digest RADseq loci using standard Illumina indexes improve deep and shallow phylogenetic resolution of Lophodermium, a widespread fungal endophyte of pine needles.

The phylogenetic and population genetic structure of symbiotic microorganisms may correlate with important ecological traits that can be difficult to directly measure, such as host preferences or dispersal rates. This study develops and tests a low-cost double-digest restriction site-associated DNA sequencing (ddRADseq) protocol to reveal among- and within-species genetic structure for Lophodermium, a genus of fungal endophytes whose evolutionary analyses have been limited by the scarcity of informative markers. The protocol avoids expensive barcoded adapters and incorporates universal indexes for multiplexing. We tested for reproducibility and functionality by comparing shared loci from sample replicates and assessed the effects of numbers of ambiguous sites and clustering thresholds on coverage depths, number of shared loci among samples, and phylogenetic reconstruction. Errors between technical replicates were minimal. Relaxing the quality-filtering criteria increased the mean coverage depth per locus and the number of loci recovered within a sample, but had little effect on the number of shared loci across samples. Increasing clustering threshold decreased the mean coverage depth per cluster and increased the number of loci recovered within a sample but also decreased the number of shared loci across samples, especially among distantly related species. The combination of low similarity clustering (70%) and relaxed quality-filtering (allowing up to 30 ambiguous sites per read) performed the best in phylogenetic analyses at both recent and deep genetic divergences. Hence, this method generated sufficient number of shared homologous loci to investigate the evolutionary relationships among divergent fungal lineages with small haploid genomes. The greater genetic resolution also revealed new structure within species that correlated with ecological traits, providing valuable insights into their cryptic life histories

Web-Based Content on Diet and Nutrition Written in Japanese: Infodemiology Study Based on Google Trends and Google Search

The increased availability of content of uncertain integrity obtained through the internet is a major concern. To date, however, there has been no comprehensive scrutiny of the fitness-for-purpose of web-based content on diet and nutrition. This cross-sectional study aims to describe diet- and nutrition-related web-based content written in Japanese, identified via a systematic extraction strategy using Google Trends and Google Search. We first identified keywords relevant for extracting web-based content (eg, blogs) on diet and nutrition written in Japanese using Google Trends. This process included identification of 638 seed terms, identification of approximately 1500 pairs of related queries (top) and search terms, the top 10% of which were extracted to identify 160 relevant pairs of related queries (top) and search terms, and identification of 107 keywords for search. We then extracted relevant web-based content using Google Search. The content (N=1703) examined here was extracted following a search based on 107 keywords. The most common themes included food and beverages (390/1703, 22.9%), weight management (366/1703, 21.49%), health benefits (261/1703, 15.33%), and healthy eating (235/1703, 13.8%). The main disseminators were information technology companies and mass media (474/1703, 27.83%), food manufacturers (246/1703, 14.45%), other (236/1703, 13.86%), and medical institutions (214/1703, 12.57%). Less than half of the content (790/1703, 46.39%) clearly indicated the involvement of editors or writers. More than half of the content (983/1703, 57.72%) was accompanied by one or more types of advertisement. The proportion of content with any type of citation reference was 40.05% (682/1703). The themes and disseminators of content were significantly associated with the involvement of editors or writers, accompaniment with advertisement, and citation of reference. In particular, content focusing on weight management was more likely to clearly indicate the involvement of editors or writers (212/366, 57.9%) and to be accompanied by advertisement (273/366, 74.6%), but less likely to have references cited (128/366, 35%). Content from medical institutions was less likely to have citation references (62/214, 29%). This study highlights concerns regarding the authorship, conflicts of interest (advertising), and the scientific credibility of web-based diet- and nutrition-related information written in Japanese. Nutrition professionals and experts should take these findings seriously because exposure to nutritional information that lacks context or seems contradictory can lead to confusion and backlash among consumers. However, more research is needed to draw firm conclusions about the accuracy and quality of web-based diet- and nutrition-related content and whether similar results can be obtained in other major mass media or social media outlets and even other languages. [Abstract copyright: ©Kentaro Murakami, Nana Shinozaki, Nana Kimoto, Hiroko Onodera, Fumi Oono, Tracy A McCaffrey, M Barbara E Livingstone, Tsuyoshi Okuhara, Mai Matsumoto, Ryoko Katagiri, Erika Ota, Tsuyoshi Chiba, Yuki Nishida, Satoshi Sasaki. Originally published in JMIR Formative Research (https://formative.jmir.org), 16.11.2023.

The UNITE database for molecular identification and taxonomic communication of fungi and other eukaryotes : sequences, taxa and classifications reconsidered

Acknowledgements We acknowledge Marie Zirk for her work in designing the UNITE logotype and creating the visual abstract for this article. Funding UNITE database development is financed by the Estonian Research Council [PRG1170]; European Union's Horizon 2020 project BGE [101059492]. The PlutoF digital infrastructure is supported by the European Union's Horizon 2020 project BiCIKL [101007492]; Estonian Research Infrastructure roadmap project DiSSCo Estonia. Funding for open access charge: UNITE Community. Conflict of interest statement. None declared.Peer reviewedPublisher PD

A confidence interval analysis of sampling effort, sequencing depth, and taxonomic resolution of fungal community ecology in the era of high-throughput sequencing

<div><p>High-throughput sequencing technology has helped microbial community ecologists explore ecological and evolutionary patterns at unprecedented scales. The benefits of a large sample size still typically outweigh that of greater sequencing depths per sample for accurate estimations of ecological inferences. However, excluding or not sequencing rare taxa may mislead the answers to the questions ‘how and why are communities different?’ This study evaluates the confidence intervals of ecological inferences from high-throughput sequencing data of foliar fungal endophytes as case studies through a range of sampling efforts, sequencing depths, and taxonomic resolutions to understand how technical and analytical practices may affect our interpretations. Increasing sampling size reliably decreased confidence intervals across multiple community comparisons. However, the effects of sequencing depths on confidence intervals depended on how rare taxa influenced the dissimilarity estimates among communities and did not significantly decrease confidence intervals for all community comparisons. A comparison of simulated communities under random drift suggests that sequencing depths are important in estimating dissimilarities between microbial communities under neutral selective processes. Confidence interval analyses reveal important biases as well as biological trends in microbial community studies that otherwise may be ignored when communities are only compared for statistically significant differences.</p></div

Effect of sequencing depths on estimates of ANOSIM R and 95% confidence intervals based on Bray-Curtis dissimilarity indices.

<p>Comparisons were made between FFE communities of a) bases and tips of <i>P</i>. <i>taeda</i> needles at 5, 10, 20 and 60 samples per group and b) between <i>P</i>. <i>torreyana</i> needles between Santa Rosa and San Diego, CA at 3, 6, 9, and 14 samples per group. Sequencing depths were tested at 100, 1000, 5000, 10000, 20000. X-axes are on a log-scale. Dotted lines indicate 95% confidence intervals for different sampling sizes. Solid lines indicate the mean. Trends for different sampling efforts were overlapped in a single plot.</p

β-diversity patterns are revealed with increasing sequencing depths between communities that differ only by random processes.

<p>Non-metric multidimensional scaling of two simulated communities drifted for 20 generations from one community with 5 million individuals and 974 OTUs (taxa) have beta-diversity patterns revealed only after 50,000 sequences per sample, which corresponds to ~1% of the community per sample. Ordinations are based on between-sample dissimilarity calculated with Bray-Curtis (left panel), Jaccard (middle panel). OTU accumulation curves (right panel) demonstrate community sampling with varying sequencing depths. From top to bottom, sequencing depths correspond to a) 100, b) 1000, c) 5000, d) 10000, and e) 50000 sequences. Insets represent the accumulation curves from the previous sequencing depth.</p

Effect of sampling effort on estimates of ANOSIM R and PERMANOVA R² effect sizes and 95% confidence intervals.

<p>Sampling effort represents random subsamples of each comparison group with replacement. ANOSIM R (grey) and PERMANOVA R² (red) values were calculated with Bray-Curtis dissimilarity and 95% confidence interval based on 1000 subsamples. a) Comparison of FFE communities between bases and tips of <i>P</i>. <i>taeda</i> needles at five-sample intervals from five to 60 samples each. b) Comparison of FFE communities between <i>P</i>. <i>torreyana</i> needles between different geographic locations (Santa Rosa and San Diego, CA) at one-sample intervals from three to 14 samples each.</p

Effect of sequencing depths on estimates of geographic community dissimilarity and 95% confidence intervals for FFE communities that differ in geography.

<p>FFE communities between nine <i>P</i>. <i>taeda</i> plots across varying distances (1–107 km) were compared. ANOSIM R (grey) and PermANOVA R² (red) values were calculated with Bray-Curtis dissimilarity and 95% confidence interval based on 1000 subsamples. Sequencing depth tested at 100, 1000, 5000, 10000, 20000. X-axes are on a log-scale. Dotted lines indicate 95% confidence interval. Solid lines indicate the mean.</p

Double-digest RADseq loci using standard Illumina indexes improve deep and shallow phylogenetic resolution of Lophodermium, a widespread fungal endophyte of pine needles.

The phylogenetic and population genetic structure of symbiotic microorganisms may correlate with important ecological traits that can be difficult to directly measure, such as host preferences or dispersal rates. This study develops and tests a low-cost double-digest restriction site-associated DNA sequencing (ddRADseq) protocol to reveal among- and within-species genetic structure for Lophodermium, a genus of fungal endophytes whose evolutionary analyses have been limited by the scarcity of informative markers. The protocol avoids expensive barcoded adapters and incorporates universal indexes for multiplexing. We tested for reproducibility and functionality by comparing shared loci from sample replicates and assessed the effects of numbers of ambiguous sites and clustering thresholds on coverage depths, number of shared loci among samples, and phylogenetic reconstruction. Errors between technical replicates were minimal. Relaxing the quality-filtering criteria increased the mean coverage depth per locus and the number of loci recovered within a sample, but had little effect on the number of shared loci across samples. Increasing clustering threshold decreased the mean coverage depth per cluster and increased the number of loci recovered within a sample but also decreased the number of shared loci across samples, especially among distantly related species. The combination of low similarity clustering (70%) and relaxed quality-filtering (allowing up to 30 ambiguous sites per read) performed the best in phylogenetic analyses at both recent and deep genetic divergences. Hence, this method generated sufficient number of shared homologous loci to investigate the evolutionary relationships among divergent fungal lineages with small haploid genomes. The greater genetic resolution also revealed new structure within species that correlated with ecological traits, providing valuable insights into their cryptic life histories