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Within microeukaryotes, genetic variation and functional variation sometimes accumulate more quickly than morphological differences. To understand the evolutionary history and ecology of such lineages, it is key to examine diversity at multiple levels of organization. In the dinoflagellate family Symbiodiniaceae, which can form endosymbioses with cnidarians ( e.g ., corals, octocorals, sea anemones, jellyfish), other marine invertebrates ( e.g. , sponges, molluscs, flatworms), and protists ( e.g ., foraminifera), molecular data have been used extensively over the past three decades to describe phenotypes and to make evolutionary and ecological inferences. Despite advances in Symbiodiniaceae genomics, a lack of consensus among researchers with respect to interpreting genetic data has slowed progress in the field and acted as a barrier to reconciling observations. Here, we identify key challenges regarding the assessment and interpretation of Symbiodiniaceae genetic diversity across three levels: species, populations, and communities. We summarize areas of agreement and highlight techniques and approaches that are broadly accepted. In areas where debate remains, we identify unresolved issues and discuss technologies and approaches that can help to fill knowledge gaps related to genetic and phenotypic diversity. We also discuss ways to stimulate progress, in particular by fostering a more inclusive and collaborative research community. We hope that this perspective will inspire and accelerate coral reef science by serving as a resource to those designing experiments, publishing research, and applying for funding related to Symbiodiniaceae and their symbiotic partnerships.

Abstract Sharks of the genus Sphyrna are under intense exploitation globally. In Brazil’s northern coast, this genus represents a high proportion of fisheries landings and comprises four species. However, due to difficulty of specific identification when specimens are landed, most of the records are limited to the genus level. Here we analyzed the effectiveness of ITS2 (Internal Transcribed Spacer 2 of rDNA) fragment length protocol (Abercrombie et al., 2005) for identifying hammerhead shark species, comparing with the analysis of COI (Cytochrome oxidase subunit I) and ITS2 sequences. We evaluated samples of muscle tissue acquired in the main fishing ports of Maranhão: Carutapera, Raposa e Tutóia. Sampling was conducted between March 2017 to March 2018 and complemented with material deposited in collection (2015). COI results indicated the occurrence of endangered species which are prohibited to be landed. These include Sphyrna mokarran (67%), S. lewini (15%), S. tudes (3%), and S. tiburo (15%). For the ITS2 marker, we investigated the optimization of the protocol developed by Abercrombie (2005) for to improve the use in this geographical area througout design of a new primers. Resumo Os tubarões do gênero Sphyrna estão sob intensa exploração em todo o mundo. No litoral norte do Brasil, esse gênero representa grande proporção dos desembarques pesqueiros e compreende quatro espécies. Porém, devido à dificuldade de identificação específica no momento do desembarque dos espécimes, a maioria dos registros limita-se ao nível do gênero. Aqui analisamos a eficácia do protocolo baseado no comprimento de fragmentos de ITS2 (Abercrombie et al., 2005) para identificar espécies de tubarão-martelo, comparando com a análise das sequências COI e ITS2. Foram avaliadas amostras de tecido muscular adquiridas nos principais portos pesqueiros do Maranhão: Carutapera, Raposa e Tutóia. A amostragem foi realizada entre março de 2017 a março de 2018 e complementada com material depositados em coleção (2015). Os resultados do COI indicaram a ocorrência de espécies ameaçadas cujo desembarque é proibido. Estes incluem Sphyrna mokarran (67%), S. lewini (15%), S. tudes (3%) e S. tiburo (15%). Para o marcador ITS2, investigamos a otimização do protocolo desenvolvido por Abercrombie (2005) para melhorar o uso nesta área geográfica através do desenho de novos primers.

is an endoparasite of fish that is found in the swim bladder of . is predominant in Asian countries, Russia and also Australia. Four species of have been found to be taxonomically valid. In India, and have been morphologically described. The sample collected from Northeast India is subjected to both morphological and molecular analysis. Molecular markers like mitochondrial Cytochrome Oxidase I gene and the ribosomal Internal Transcribed Spacer 2 gene markers have aided the identification and characterization of parasites particularly flatworms. The morphological and the molecular assessment indicate the presence of in Northeast India. This report is the first locality and regional report from India.

Dendranthema indicum var. aromaticum, which is an aromatic plant with a strong and special fragrance throughout the whole plant, is used for the treatment of colds and headaches, and as a mosquito repellant in Shennongjia, Hubei province, China. To analyze the composition of the essential oil from this medicinal herb, we developed a gas chromatography-mass Spectrometry (GC-MS) method including microwave-assisted extraction, hydrodistillation and direct headspace analysis in two different stationary phase columns. In total, 115 volatile compounds were identified, of which 90 compounds were identified using Rxi-5MS and 78 using HP-INNOWAX. Our results revealed that the oil was mainly composed of five categories of compound: oxygenated monoterpenes (28.76–78.10%), oxygenated sesquiterpenes (4.27–38.06%), sesquiterpenes (3.22–11.57%), fatty hydrocarbons (1.65–9.81%) and monoterpenes (0–3.32%). The major constituents are α-thujone, β-thujone, cis-sabinol, sabinyl acetate and (-)-neointermedeol.However, the essential oil composition in the published literature differs significantly. Therefore, a cluster analysis was carried out using the top ten compositions in the reported literature as well as this study, using Minitab software. To provide detailed information on plant origin, the ITS1-5.8s-ITS2 region was amplified and sequenced (Accession No. MF668250). Besides, in order to provide a macroscopic view of the chemical composition, the biosynthetic pathway of the main components was summarized according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and the published literatures.

Abstract Melting snow and glacier surfaces host microalgal blooms in polar and mountainous regions. The aim of this study was to determine the dominant taxa at the species level in the European Arctic and the Alps. A standardized protocol for amplicon metabarcoding using the 18S rRNA gene and ITS2 markers was developed. This is important because previous biodiversity studies have been hampered by the dominance of closely related algal taxa in snow and ice. Due to the limited resolution of partial 18S rRNA Illumina sequences, the hypervariable ITS2 region was used to further discriminate between the genotypes. Our results show that red snow was caused by the cosmopolitan Sanguina nivaloides (Chlamydomonadales, Chlorophyta) and two as of yet undescribed Sanguina species. Arctic orange snow was dominated by S. aurantia, which was not found in the Alps. On glaciers, at least three Ancylonema species (Zygnematales, Streptophyta) dominated. Golden-brown blooms consisted of Hydrurus spp. (Hydrurales, Stramenophiles) and these were mainly an Arctic phenomenon. For chrysophytes, only the 18S rRNA gene but not ITS2 sequences were amplified, showcasing how delicate the selection of eukaryotic ‘universal’ primers for community studies is and that primer specificity will affect diversity results dramatically. We propose our approach as a ‘best practice’. Communities of psychrophilic microalgae causing blooms in melting snow and on glaciers in Europe and the European Arctic were evaluated using optimized molecular primers.

Aim Rapid and accurate species identification is the foundation for biodiversity assessment. DNA barcoding has been shown to be an effective tool to overcome the taxonomic impediment to facilitate biodiversity conservation in temperate forests. However, this tool has rarely been considered for use in tropical forests. This study aims to investigate the utility and species resolution of DNA barcoding in a subtropical region. Location The Dinghushan National Nature Reserve (DNNR) in China. Methods A DNA barcoding database was constructed for 531 trees present in the DNNR. We used a phylogenetic method (neighbour-joining trees) and sequence similarity (all-to-all BLASTn searches) to evaluate the utility and species resolution of five DNA barcode regions (rbcL, matK, ITS, ITS2 and trnH-psbA), both singly and in combinations of two or three region. Results The combination of rbcL + matK + ITS had the highest species resolution (94.19%). However, when considering the difficulty of sequence recoverability, rbcL + ITS2 performed best (64.64%). Species resolution for large genera containing more than two species was substantially lower than that for small genera with one and two species per genus. Local small spatial scales (1-ha quadrats) resulted in moderately improved species resolution (70.82% for rbcL + ITS2) compared to larger spatial scales (20 and 1133 ha). We document incongruent signals between nuclear and cpDNA regions and the challenges associated with barcoding large genera inherent to subtropical floras. Main conclusions This study considerably expands the global DNA barcode database for subtropical trees. Based on cost-effectiveness and the trade-off between sequence recovery and species resolution, we suggest that the rbcL + ITS2 barcode combination is an effective tool for documenting plant diversity in the DNNR. This study also sheds some light on the limitations and challenges for the application of barcoding to conservation biogeography in subtropical forests.

Background Most studies describing the human gut microbiome in healthy and diseased states have emphasized the bacterial component, but the fungal microbiome (i.e., the mycobiome) is beginning to gain recognition as a fundamental part of our microbiome. To date, human gut mycobiome studies have primarily been disease centric or in small cohorts of healthy individuals. To contribute to existing knowledge of the human mycobiome, we investigated the gut mycobiome of the Human Microbiome Project (HMP) cohort by sequencing the Internal Transcribed Spacer 2 (ITS2) region as well as the 18S rRNA gene. Results Three hundred seventeen HMP stool samples were analyzed by ITS2 sequencing. Fecal fungal diversity was significantly lower in comparison to bacterial diversity. Yeast dominated the samples, comprising eight of the top 15 most abundant genera. Specifically, fungal communities were characterized by a high prevalence of Saccharomyces , Malassezia , and Candida , with S. cerevisiae , M. restricta , and C. albicans operational taxonomic units (OTUs) present in 96.8, 88.3, and 80.8% of samples, respectively. There was a high degree of inter- and intra-volunteer variability in fungal communities. However, S. cerevisiae , M. restricta , and C. albicans OTUs were found in 92.2, 78.3, and 63.6% of volunteers, respectively, in all samples donated over an approximately 1-year period. Metagenomic and 18S rRNA gene sequencing data agreed with ITS2 results; however, ITS2 sequencing provided greater resolution of the relatively low abundance mycobiome constituents. Conclusions Compared to bacterial communities, the human gut mycobiome is low in diversity and dominated by yeast including Saccharomyces , Malassezia , and Candida . Both inter- and intra-volunteer variability in the HMP cohort were high, revealing that unlike bacterial communities, an individual’s mycobiome is no more similar to itself over time than to another person’s. Nonetheless, several fungal species persisted across a majority of samples, evidence that a core gut mycobiome may exist. ITS2 sequencing data provided greater resolution of the mycobiome membership compared to metagenomic and 18S rRNA gene sequencing data, suggesting that it is a more sensitive method for studying the mycobiome of stool samples.

Seed microbiome includes special endophytic or epiphytic microbial taxa associated with seeds, which affects seed germination, plant growth, and health. Here, we analyzed the core microbiome of 21 Salvia miltiorrhiza seeds from seven different geographic origins using 16S rDNA and ITS amplicon sequencing, followed by bioinformatics analysis. The whole bacterial microbiome was classified into 17 microbial phyla and 39 classes. Gammaproteobacteria (67.6%), Alphaproteobacteria (15.6%), Betaproteobacteria (2.6%), Sphingobacteria (5.0%), Bacilli (4.6%), and Actinobacteria (2.9%) belonged to the core bacterial microbiome. Dothideomycetes comprised 94% of core fungal microbiome in S. miltiorrhiza seeds, and another two dominant classes were Leotiomycetes (3.0%) and Tremellomycetes (2.0%). We found that terpenoid backbone biosynthesis, degradation of limonene, pinene, and geraniol, and prenyltransferases, were overrepresented in the core bacterial microbiome using phylogenetic examination of communities by reconstruction of unobserved states (PICRUSt) software. We also found that the bacterial genera Pantoea, Pseudomonas, and Sphingomonas were enriched core taxa and overlapped among S. miltiorrhiza, maize, bean, and rice, while a fungal genus, Alternaria, was shared within S. miltiorrhiza, bean, and Brassicaceae families. These findings highlight that seed-associated microbiomeis an important component of plant microbiomes, which may be a gene reservoir for secondary metabolism in medicinal plants.

Background Microorganisms serve important functions within numerous eukaryotic host organisms. An understanding of the variation in the plant niche-level microbiome, from rhizosphere soils to plant canopies, is imperative to gain a better understanding of how both the structural and functional processes of microbiomes impact the health of the overall plant holobiome. Using Populus trees as a model ecosystem, we characterized the archaeal/bacterial and fungal microbiome across 30 different tissue-level niches within replicated Populus deltoides and hybrid Populus trichocarpa × deltoides individuals using 16S and ITS2 rRNA gene analyses. Results Our analyses indicate that archaeal/bacterial and fungal microbiomes varied primarily across broader plant habitat classes (leaves, stems, roots, soils) regardless of plant genotype, except for fungal communities within leaf niches, which were greatly impacted by the host genotype. Differences between tree genotypes are evident in the elevated presence of two potential fungal pathogens, Marssonina brunnea and Septoria sp., on hybrid P. trichocarpa × deltoides trees which may in turn be contributing to divergence in overall microbiome composition. Archaeal/bacterial diversity increased from leaves, to stem, to root, and to soil habitats, whereas fungal diversity was the greatest in stems and soils. Conclusions This study provides a holistic understanding of microbiome structure within a bioenergy relevant plant host, one of the most complete niche-level analyses of any plant. As such, it constitutes a detailed atlas or map for further hypothesis testing on the significance of individual microbial taxa within specific niches and habitats of Populus and a baseline for comparisons to other plant species.

The ubiquity of plastic debris in marine environments raises the question, what impacts do plastics have on our marine microbiota? To investigate this, we applied bacterial 16S rRNA gene and fungal ITS2 region sequencing to identify changes in microbial biofilm community compositions on marine plastic, over time. We sampled biofilm on virgin linear low-density polyethylene (LLDPE), nylon-6 (PA) and glass after 2, 6 and 12 weeks of constant immersion in Te Whakaraupō-Lyttelton Harbour, Aotearoa-New Zealand. Of the prokaryotes, Proteobacteria and Bacteroidetes were predominant in all samples and Verrucomicrobiota were most abundant in mature biofilms. Microbial communities on the three substrate types were significantly distinct from those in the surrounding seawater, regardless of age, but not between attachment substrates. Bacterial communities occurring two weeks after immersion and fungal communities at six weeks were found to vary more among substrate types than at other times; however, no significant substrate-specific communities were identified overall. Taxa closely related to previously reported plastic-biodegrading species were found in very low abundance across all substrates, including on the glass slides. Our findings suggest that microorganisms do not selectively persist on the LLDPE or PA surfaces to gain significant direct metabolic benefit, instead using these plastics primarily as an attachment surface on which they form generalist biofilm communities.