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1. TheOrganelle in theOintment: CrypticMitochondriaBiasCross-SpeciesMicrobiomeComparisons

   Dylan Sonett, Tanya Brown ( May 2022 , International Symposium on New Frontiers in Reef Coral Biotechnology (5 May 2022, Taiwan))

   The microbiomes of tropical corals are actively studied using 16S rRNA gene amplicons to understand microbial roles in coral health, metabolism, and disease resistance. However, due to the prokaryotic origins of mitochondria, primers targeting bacterial and archaeal 16S rRNA genes may also amplify homologous 12S mitochondrial rRNA genes from the host coral, associated microbial eukaryotes, and encrusting organisms. Standard microbial bioinformatics pipelines attempt to identify and remove these sequences by comparing them to reference taxonomies. However, commonly used tools have severely under-annotated mitochondrial sequences in 1440 coral microbiomes from the Global Coral Microbiome Project, preventing annotation of over 95% of reads in some samples. This issue persists when using Greengenes or SILVA prokaryotic reference taxonomies, and in other hosts, including 16S studies of vertebrates, and of marine sponges. Worse, mitochondrial under-annotation varies between coral families and across coral compartments, biasing comparisons of  - and  -diversity. By supplementing existing reference taxonomies with over 3000 animal mitochondrial rRNA gene sequences, we resolved roughly 97% of unique unclassified sequences as mitochondrial. These additional sequences did not cause a false elevation in mitochondrial annotations in mock communities with known compositions. We recommend using these extended taxonomies for coral microbiome analysis and whenever eukaryotic contamination may be a concern. 

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2. Shifts in the coral microbiome in response to in situ experimental deoxygenation

   https://doi.org/10.1128/aem.00577-23  

   Howard, Rachel D. ; Schul, Monica D. ; Rodriguez Bravo, Lucia M. ; Altieri, Andrew H. ; Meyer, Julie L. ( November 2023 , Applied and Environmental Microbiology)

   Biddle, Jennifer F. (Ed.)

   Global climate change impacts marine ecosystems through rising surface temperatures, ocean acidification, and deoxygenation. While the response of the coral holobiont to the first two effects has been relatively well studied, less is known about the response of the coral microbiome to deoxygenation. In this study, we investigated the response of the microbiome to hypoxia in two coral species that differ in their tolerance to hypoxia. We conductedin situoxygen manipulations on a coral reef in Bahía Almirante on the Caribbean coast of Panama, which has previously experienced documented episodes of hypoxia. Naïve coral colonies (previously unexposed to hypoxia) ofSiderastrea sidereaandAgaricia lamarckiwere transplanted to a reef and either enclosed in chambers that created hypoxic conditions or left at ambient oxygen levels. We collected samples of surface mucus and tissue after 48 hours of exposure and characterized the microbiome by sequencing 16S rRNA genes. We found that the microbiomes of the two coral species were distinct from one another and remained so after exhibiting similar shifts in microbiome composition in response to hypoxia. There was an increase in both abundance and number of taxa of anaerobic microbes after exposure to hypoxia. Some of these taxa may play beneficial roles in the coral holobiont by detoxifying the surrounding environment during hypoxic stress or may represent opportunists exploiting host stress. This work describes the first characterization of the coral microbiome under hypoxia and is an initial step toward identifying potential beneficial bacteria for corals facing this environmental stressor.

   Marine hypoxia is a threat for corals but has remained understudied in tropical regions where coral reefs are abundant. Though microbial symbioses can alleviate the effects of ecological stress, we do not yet understand the taxonomic or functional response of the coral microbiome to hypoxia. In this study, we experimentally lowered oxygen levels aroundSiderastrea sidereaandAgaricia lamarckicoloniesin situto observe changes in the coral microbiome in response to deoxygenation. Our results show that hypoxia triggers a stochastic change of the microbiome overall, with some bacterial families changing deterministically after just 48 hours of exposure. These families represent an increase in anaerobic and opportunistic taxa in the microbiomes of both coral species. Thus, marine deoxygenation destabilizes the coral microbiome and increases bacterial opportunism. This work provides novel and fundamental knowledge of the microbial response in coral during hypoxia and may provide insight into holobiont function during stress.

    

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3. DEPP: Deep Learning Enables Extending Species Trees using Single Genes

   https://doi.org/10.1093/sysbio/syac031  

   Jiang, Yueyu ; Balaban, Metin ; Zhu, Qiyun ; Mirarab, Siavash ; Solis-Lemus, ed., Claudia ( April 2022 , Systematic Biology)

   Placing new sequences onto reference phylogenies is increasingly used for analyzing environmental samples, especially microbiomes. Existing placement methods assume that query sequences have evolved under specific models directly on the reference phylogeny. For example, they assume single-gene data (e.g., 16S rRNA amplicons) have evolved under the GTR model on a gene tree. Placement, however, often has a more ambitious goal: extending a (genome-wide) species tree given data from individual genes without knowing the evolutionary model. Addressing this challenging problem requires new directions. Here, we introduce Deep-learning Enabled Phylogenetic Placement (DEPP), an algorithm that learns to extend species trees using single genes without prespecified models. In simulations and on real data, we show that DEPP can match the accuracy of model-based methods without any prior knowledge of the model. We also show that DEPP can update the multilocus microbial tree-of-life with single genes with high accuracy. We further demonstrate that DEPP can combine 16S and metagenomic data onto a single tree, enabling community structure analyses that take advantage of both sources of data. [Deep learning; gene tree discordance; metagenomics; microbiome analyses; neural networks; phylogenetic placement.]

    

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4. Evaluating and Improving Small Subunit rRNA PCR Primer Coverage for Bacteria, Archaea, and Eukaryotes Using Metagenomes from Global Ocean Surveys

   https://doi.org/10.1128/mSystems.00565-21  

   McNichol, Jesse ; Berube, Paul M. ; Biller, Steven J. ; Fuhrman, Jed A. ( June 2021 , mSystems)

   Gilbert, Jack A. (Ed.)

   ABSTRACT Small subunit rRNA (SSU rRNA) amplicon sequencing can quantitatively and comprehensively profile natural microbiomes, representing a critically important tool for studying diverse global ecosystems. However, results will only be accurate if PCR primers perfectly match the rRNA of all organisms present. To evaluate how well marine microorganisms across all 3 domains are detected by this method, we compared commonly used primers with >300 million rRNA gene sequences retrieved from globally distributed marine metagenomes. The best-performing primers compared to 16S rRNA of bacteria and archaea were 515Y/926R and 515Y/806RB, which perfectly matched over 96% of all sequences. Considering cyanobacterial and chloroplast 16S rRNA, 515Y/926R had the highest coverage (99%), making this set ideal for quantifying marine primary producers. For eukaryotic 18S rRNA sequences, 515Y/926R also performed best (88%), followed by V4R/V4RB (18S rRNA specific; 82%)—demonstrating that the 515Y/926R combination performs best overall for all 3 domains. Using Atlantic and Pacific Ocean samples, we demonstrate high correspondence between 515Y/926R amplicon abundances (generated for this study) and metagenomic 16S rRNA (median R 2 = 0.98, n  = 272), indicating amplicons can produce equally accurate community composition data compared with shotgun metagenomics. Our analysis also revealed that expected performance of all primer sets could be improved with minor modifications, pointing toward a nearly completely universal primer set that could accurately quantify biogeochemically important taxa in ecosystems ranging from the deep sea to the surface. In addition, our reproducible bioinformatic workflow can guide microbiome researchers studying different ecosystems or human health to similarly improve existing primers and generate more accurate quantitative amplicon data. IMPORTANCE PCR amplification and sequencing of marker genes is a low-cost technique for monitoring prokaryotic and eukaryotic microbial communities across space and time but will work optimally only if environmental organisms match PCR primer sequences exactly. In this study, we evaluated how well primers match globally distributed short-read oceanic metagenomes. Our results demonstrate that primer sets vary widely in performance, and that at least for marine systems, rRNA amplicon data from some primers lack significant biases compared to metagenomes. We also show that it is theoretically possible to create a nearly universal primer set for diverse saline environments by defining a specific mixture of a few dozen oligonucleotides, and present a software pipeline that can guide rational design of primers for any environment with available meta’omic data. 

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5. Community structure of coral microbiomes is dependent on host morphology

   https://doi.org/10.1186/s40168-022-01308-w  

   Morrow, Kathleen M. ; Pankey, M. Sabrina ; Lesser, Michael P. ( December 2022 , Microbiome)

   Abstract Background The importance of symbiosis has long been recognized on coral reefs, where the photosynthetic dinoflagellates of corals (Symbiodiniaceae) are the primary symbiont. Numerous studies have now shown that a diverse assemblage of prokaryotes also make-up part of the microbiome of corals. A subset of these prokaryotes is capable of fixing nitrogen, known as diazotrophs, and is also present in the microbiome of scleractinian corals where they have been shown to supplement the holobiont nitrogen budget. Here, an analysis of the microbiomes of 16 coral species collected from Australia, Curaçao, and Hawai’i using three different marker genes (16S rRNA, nif H, and ITS2) is presented. These data were used to examine the effects of biogeography, coral traits, and ecological life history characteristics on the composition and diversity of the microbiome in corals and their diazotrophic communities. Results The prokaryotic microbiome community composition (i.e., beta diversity) based on the 16S rRNA gene varied between sites and ecological life history characteristics, but coral morphology was the most significant factor affecting the microbiome of the corals studied. For 15 of the corals studied, only two species Pocillopora acuta and Seriotopora hystrix , both brooders, showed a weak relationship between the 16S rRNA gene community structure and the diazotrophic members of the microbiome using the nif H marker gene, suggesting that many corals support a microbiome with diazotrophic capabilities. The order Rhizobiales , a taxon that contains primarily diazotrophs, are common members of the coral microbiome and were eight times greater in relative abundances in Hawai’i compared to corals from either Curacao or Australia. However, for the diazotrophic component of the coral microbiome, only host species significantly influenced the composition and diversity of the community. Conclusions The roles and interactions between members of the coral holobiont are still not well understood, especially critical functions provided by the coral microbiome (e.g., nitrogen fixation), and the variation of these functions across species. The findings presented here show the significant effect of morphology, a coral “super trait,” on the overall community structure of the microbiome in corals and that there is a strong association of the diazotrophic community within the microbiome of corals. However, the underlying coral traits linking the effects of host species on diazotrophic communities remain unknown. 

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