More Like this

1. Comprehensive single‐PCR 16S and 18S rRNA community analysis validated with mock communities, and estimation of sequencing bias against 18S

   https://doi.org/10.1111/1462-2920.15553  

   Yeh, Yi‐Chun; McNichol, Jesse; Needham, David_M; Fichot, Erin_B; Berdjeb, Lyria; Fuhrman, Jed_A (May 2021, Environmental Microbiology)

   Summary Universal primers for SSU rRNA genes allow profiling of natural communities by simultaneously amplifying templates from Bacteria, Archaea, and Eukaryota in a single PCR reaction. Despite the potential to show relative abundance for all rRNA genes, universal primers are rarely used, due to various concerns including amplicon length variation and its effect on bioinformatic pipelines. We thus developed 16S and 18S rRNA mock communities and a bioinformatic pipeline to validate this approach. Using these mocks, we show that universal primers (515Y/926R) outperformed eukaryote‐specific V4 primers in observed versus expected abundance correlations (slope = 0.88 vs. 0.67–0.79), and mock community members with single mismatches to the primer were strongly underestimated (threefold to eightfold). Using field samples, both primers yielded similar 18S beta‐diversity patterns (Mantel test,p < 0.001) but differences in relative proportions of many rarer taxa. To test for length biases, we mixed mock communities (16S + 18S) before PCR and found a twofold underestimation of 18S sequences due to sequencing bias. Correcting for the twofold underestimation, we estimate that, in Southern California field samples (1.2–80 μm), there were averages of 35% 18S, 28% chloroplast 16S, and 37% prokaryote 16S rRNA genes. These data demonstrate the potential for universal primers to generate comprehensive microbiome profiles. 

   more » « less
2. Ecological succession of the sponge cryptofauna in Hawaiian reefs add new insights to detritus production by pioneering species

   https://doi.org/10.1038/s41598-022-18856-8  

   Vicente, Jan; Timmers, Molly A.; Webb, Maryann K.; Bahr, Keisha D.; Jury, Christopher P.; Toonen, Robert J. (December 2022, Scientific Reports)

   Abstract Successional theory proposes that fast growing and well dispersed opportunistic species are the first to occupy available space. However, these pioneering species have relatively short life cycles and are eventually outcompeted by species that tend to be longer-lived and have lower dispersal capabilities. Using Autonomous Reef Monitoring Structures (ARMS) as standardized habitats, we examine the assembly and stages of ecological succession among sponge species with distinctive life history traits and physiologies found on cryptic coral reef habitats of Kāneʻohe Bay, Hawaiʻi. Sponge recruitment was monitored bimonthly over 2 years on ARMS deployed within a natural coral reef habitat resembling the surrounding climax community and on ARMS placed in unestablished mesocosms receiving unfiltered seawater directly from the natural reef deployment site. Fast growing haplosclerid and calcareous sponges initially recruited to and dominated the mesocosm ARMS. In contrast, only slow growing long-lived species initially recruited to the reef ARMS, suggesting that despite available space, the stage of ecological succession in the surrounding habitat influences sponge community development in uninhabited space. Sponge composition and diversity between early summer and winter months within mesocosm ARMS shifted significantly as the initially recruited short-lived calcareous and haplosclerid species initially recruit and then died off. The particulate organic carbon contribution of dead sponge tissue from this high degree of competition-free community turnover suggests a possible new component to the sponge loop hypothesis which remains to be tested among these pioneering species. This source of detritus could be significant in early community development of young coastal habitats but less so on established coral reefs where the community is dominated by long-lived colonial sponges. 

   more » « less
3. 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. 

   more » « less
4. Community structure and microhabitat associations of cryptobenthic reef fishes in Veracruz, Mexico

   https://doi.org/10.1007/s00338-025-02763-1  

   Rivera-Higueras, Mariana; Hemingson, Christopher R; Pouchoulen_Alemán, Aurora_de Monserrat; Cipolla, Kyra_Jean M; Souza, Philip M; Brandl, Simon J (October 2025, Coral Reefs)

   Local habitat availability can strongly affect animal communities. On coral reefs, the biodiversity of small, bottom-dwelling (‘cryptobenthic’) reef fishes and drivers of their community assembly have yet to be explored in many locations. Here, we investigate how local and regional factors shape the structure and composition of cryptobenthic reef fish communities in the Veracruz Reef System National Park (VRS) in the Gulf of Mexico (GoM). Focusing on five reefs in the VRS, we surveyed cryptobenthic reef fish communities at scales of reef outcrops (~ 3–5 m2) and isolated microhabitats, while also quantifying the benthic composition of each reef to determine microhabitat availability. We found no significant differences in species richness or abundance across park regions and reef zones, but community composition differed qualitatively across reef zones. Furthermore, we discovered strong differences in cryptobenthic reef fishes’ preferences for various microhabitats, which are likely to drive community assembly and provide evidence for species-specific vulnerabilities to reef degradation. Caves harbored the highest biodiversity and abundance of cryptobenthic fishes, while gorgonian soft corals and algae supported the fewest species and individuals. The endemic gobies Tigrigobius redimiculus and Elacatinus jarocho both showed high abundance and occurrence but displayed opposite patterns of microhabitat specialization; T. redimiculus was categorized as a microhabitat generalist, while E. jarocho was revealed as a cave-dwelling specialist species. Overall, our quantitative exploration of the cryptobenthic reef fish community in the southwest GoM provides a crucial baseline for habitat and biodiversity monitoring in the region and highlights E. jarocho as an emblematic, endemic indicator species that will be vulnerable to extinction if further reduction of habitat complexity occurs. 

   more » « less
5. DNA metabarcoding captures different macroinvertebrate biodiversity than morphological identification approaches across a continental scale

   https://doi.org/10.1002/edn3.453  

   Emmons, Sean C.; Compson, Zacchaeus G.; Malish, Megan C.; Busch, Michelle H.; Saenz, Veronica; Higgins, Kierstyn T.; Allen, Daniel C. (July 2023, Environmental DNA)

   Abstract DNA‐based aquatic biomonitoring methods show promise to provide rapid, standardized, and efficient biodiversity assessment to supplement and in some cases replace current morphology‐based approaches that are often less efficient and can produce inconsistent results. Despite this potential, broad‐scale adoption of DNA‐based approaches by end‐users remains limited, and studies on how these two approaches differ in detecting aquatic biodiversity across large spatial scales are lacking. Here, we present a comparison of DNA metabarcoding and morphological identification, leveraging national‐scale, open‐source, ecological datasets from the National Ecological Observatory Network (NEON). Across 24 wadeable streams in North America with 179 paired sample comparisons, we found that DNA metabarcoding detected twice as many unique taxa than morphological identification overall. The two approaches showed poor congruence in detecting the same taxa, averaging 59%, 35%, and 23% of shared taxa detected at the order, family, and genus levels, respectively. Importantly, the two approaches detected different proportions of indicator taxa like %EPT and %Chironomidae. DNA metabarcoding detected far fewer Chironomid and Trichopteran taxa than morphological identification, but more Ephemeropteran and Plecopteran taxa, a result likely due to primer choice. Overall, our results showed that DNA metabarcoding and morphological identification detected different benthic macroinvertebrate communities. Despite these differences, we found that the same environmental variables were correlated with invertebrate community structure, suggesting that both approaches can accurately detect biodiversity patterns across environmental gradients. Further refinement of DNA metabarcoding protocols, primers, and reference libraries–as well as more standardized, large‐scale comparative studies–may improve our understanding of the taxonomic agreement and data linkages between DNA metabarcoding and morphological approaches. 

   more » « less