Quantification of gene copy numbers is valuable in marine microbial ecology: A comment to Meiler et al. (2022)

Marine nitrogen (N2) fixation is important in the global biogeochemical cycling of N, and interlinked carbon (C). Understanding predicting marine N2 requires information on diazotroph species specific rates growth fixation, their biogeography, physiology nutrient limitation diazotrophs nondiazotrophs (for basis competition models). Because many are not easily visualized or quantified yet uncultivated, application polymerase chain reaction (PCR) methods to amplify nifH gene, which encodes nitrogenase, enzyme that catalyzes have led multiple discoveries including new microorganisms (Zehr et al. 1998; Zehr Capone 2020). Moreover, use quantitative PCR (qPCR) has revealed unexpected biogeography key regionally globally (Church 2005; Bentzon-Tilia 2015; Langlois Messer Shiozaki 2017; Harding 2018; Mulholland 2019). Biogeochemical modeling approaches also been invaluable for providing hypothetical dynamic biomass associated with size classes based a number assumptions (e.g., growth, uptake characteristics, mortality; Dutkiewicz 2014). It is, however, difficult validate these models since there few comprehensive datasets N2-fixing organism biogeography. In recent study, Meiler (2022) attempted assembled qPCR data deduce cyanobacterial using derived currency cell abundances biomass, order assess value validating numerical model Using published studies (six publications varied used different approaches) had reported situ gene abundances, numbers (e. g. Hynes 2012; Foster 2013; Krupke White 2018) they conversion factors from numbers, cells biomass. The maximum minimum range (i.e., extremes, average values) both steps were then applied an existing database (Luo Tang Cassar This approach generated ranges depth integrated abundance inferred counts across oceanic regions latitudinal gradients comparison output. At biome level, predicted measurements was approximately four orders magnitude greater than dissimilarity output simulations same group over period 6 years. concluded error converting “currency” (form units variables needed by models) too large utility validation (ranging Trichodesmium one Crocosphaera, UCYN-B). They likely other genes, best as indicator presence/absence rather measure evaluating models. Although analysis commonly very useful highlights need further research, conclusion “Despite its usefulness diazotrophic presence, may only weakly correlate biomass” worth revisiting. We disagree correlated because select (1) about taxa classes, (2) inflated significance polyploidy undermining quantitation diazotrophs, (3) resulting unnecessarily high combination two values metrics, (4) original reveal spatial patterns consistent direct indirect observations rate contradicting assertion assessment. argue at root disconnect between environmental measured outputs, additional affect (or any molecular imaging methods) One factor defined genera bridge physiological characteristics size. define up five 3 15 μm diameter (Dutkiewicz 2021). treated Trichodesmium, UCYN-A symbiosis, diatoms Richelia symbionts, Crocosphaera (some form aggregates), vary (Pierella Karlusich primers generally target narrowly taxonomic group, thus can distinguish abundant rare taxa, sizes biogeographic ranges. However, combined (qPCR data) within genus size, but frequency occurrence oceans groups addressed each own challenges quantification. spp. filamentous, cyanobacteria do specialized heterocyst 2023). greatly (6–16 width 4–23 long diverse species; 2012), (Webb 2009, 2023; typically found wide areas ocean. Filamentous heterocyst-forming physical location symbiont, filaments per diatom, heterocysts filament, degree aggregation formation (Caputo (Candidatus Atelocyanobacterium thalassa) collection mostly uncultivated strains symbionts metabolically reduced cyanobacterium UCYN-A, endosymbiotic haptophyte Braarudosphaera bigelowii relatives (Hagino Farnelid 2016). There coastal open ocean ecotypes least 5 (Turk-Kubo watsonii planktonic comprised closely related 2009; 2023), include free-living symbiotic forms, aggregation. C. natural populations small forms aggregates Most common Bench Hence, illustrated here (see below), fundamental problem when defining species, all binned (2022). inevitably contributes variability calculated : C symbioses, will make copy highly questionable. Richelia/Calothrix, (2019) strains/species unfortunately contributed factors. conclusions well some (Krupke Wilson calculate gene-to-cell major taxa-specific nitrogenase genes (nifH) abundances. differed used, location. among available attempt desired factors, adequate this purpose due limitations lead erroneous regarding values. cited determine nifH-to-cell conversions: (2018) microscopy vs. qPCR; (2017) flow cytometry sorted coupled (2013) catalyzed reporter hybridization (CARD-FISH) qPCR. These sometimes involved analyses separate sample collections, noted (2022), be problematic drawn. For instance, free (puffs tufts) buoyant float sink notoriously heterogeneously distributed (Rodier Borgne 2008). Therefore, measurement comparisons made exactly water sample, replicates CTD cast replicate casts station. Nonetheless, study ranged 2 (130 ± 239 cell; 2018), compared total propagation 1.4–1405 cell. Furthermore, types, such size-fractionations. example, samples (pooled FISH filter fractions compare 0.2-μm filtered DNA samples) CARD-FISH qPCR, respectively, involving another source purposes. A shows well-correlated, certainly varying (Fig. 1, panels A-D, Gradoville 2022). taken species/strains known hosts), study. individual UCYN-A1 UCYN-A2, differ (Farnelid 2016; Turk-Kubo 2021) represent address Similarly, diatom symbiont combined, despite fact probes symbioses physiology, case sources C, associations several sizes, differing filament 2021; Flores variety issues pertain quantifying (DNA) measurement. Since targeted UCYN-A1), precise estimates extraction efficiencies relatively constant species. efficiency issue approach, metagenomics 2021), so implications unique Polyploidy, genome copies cell, (and eukaryotes), recently recognized respect (Sargent Pierella little known, studies, potential significant. even field suggest could modeled linear relationship 2). Thus, more straightforward until would simple representative exemplified we advocate extreme caution should number, cases necessary desirable below). example provided Figs. show Rather, find choice metric amplifies problems specifically se apply counts). cell-to-biomass calculations perhaps most convert (in units), (Foster 2011; parameters (Strathman 1967; Verity 1992). estimated sensitive assumed dimensions, elemental stoichiometry. Only directly cultures Much came designed universal estimating isotope enrichment labeled N nanoscale secondary ion mass spectrometry (nanoSIMS; 2018). nanoSIMS, partially analyzed beam ionizes successive layers dependent orientation fraction “burned” through. Total calculating volumes 3D shapes two-dimensional slice data. nanoSIMS tracer experimental analysis, accurate cellular better cultivated organisms and/or techniques. chosen extremes (the Arctic; particularly focus conversions amplified combining differences strains, though strain inhabits ocean, UCYN-A1, much smaller others constrained host, relevant 2–3 diameter, whereas UCYN-A2 times (Cabello typical models, independent method derive (including cytometry). data, general challenge seek extrapolate impact larger scales, (Coles 2017). describe variance RNA) methods. Combined handling metric, leave casual reader impression flawed technique no value. allow if stress demonstrated seasonality Cabello 2019), (Shiozaki Hallstrøm 2022) hotspots blooms detect (Goebel 2008; Moisander 2010). insights into drivers now importance cycling. Also, it possible sorting sequencing (Bombar current allowing quantification sea, critical obtaining given temporal scales patchiness populations. Finally, examine dataset fact, match echo distribution compilations observations, inverse distributions, ecosystem Aumont Landolfi Letscher Wang Indeed, Luo (2012) while lacking newer compilation, demonstrates close (their fig. 8a) converted 9a). main extrapolating (qPCR, applies metagenomic biomasses organisms. point, relationships poorly supported extrema evaluate empirical inappropriate unintentionally support poor understanding controls diazotrophy. Taken together, obtained variable sampling strategies unsuited gene: cell: relationships. confidence backbone limited, makes restricted questionable; emphasizes ranges, although reasonable averages extremes; basic premise integrating goal. summary, First, combines errors measures those second error, ranging does applications virtually oceanographic discussion leads implication observed, extant agree what More generally, reflect content, microbial modeling, re-evaluated. conclusion, thought-provoking insights, nifH, valuable. encourage continued enumeration gain controls, ecology microbes oceans. authors thank Kendra Turk-Kubo, Mary R. Gradoville, Victoria J. Coles, Rachel Foster, Annette Hynes, Eric Webb, Tracy Villareal, Sam Wilson, Angelicque suggestions insights. J.Z. Simons Foundation (SCOPE #724220 Life Sciences #824082). L.R. grant 0217-00089B Independent Research Fund Denmark. declare conflict interest publication article.