The Critical Importance of Rhodoliths in the Life Cycle Completion of Both Macro- and Microalgae, and as Holobionts for the Establishment and Maintenance of Marine Biodiversity

Abstract

Rhodoliths are the main hard substrata for the attachment of benthic macroalgae in the NW Gulf of Mexico rubble habitats that are associated with salt domes, unique deep bank habitats at ∼50-90 m depth on the continental shelf offshore Louisiana and Texas. With the advent of additional sequencing technologies, methodologies for biodiversity assessments are now rapidly shifting to DNA metabarcoding, i.e., High Throughput Sequencing (HTS) of environmental DNA mixtures with standardized molecular markers, such as 16S V4, for rapid, cost-effective biodiversity measurement. We newly tested 16S V4 metabarcoding on endolithic portions of mesophtic rhodoliths exhibiting low phototroph colonization that revealed a hidden, cryptic algal diversity targeting spores, propagules, and unsuspected life history stages. We explored cryo-SEM as a potentially more informative method than regular SEM to minimize artifacts of sample preparation in the study of endolithic cell inclusions which brought to light a suite of microalgal stages. We were able to differentiate floridean starch from cellular inclusions. We associated the effect of anatomical growth pattern on presence or absence of cellular inclusions in biogenic rhodoliths. Analyses of combined 16S V4 metabarcodes and 16S Sanger sequences of two red algal orders, the Halymeniales and Bonnemaisoniales, increased the established record of diversity in the region. We view rhodoliths as marine biodiversity hotspots that may function as seedbanks, temporary reservoirs for life history stages of ecologically important eukaryotic microalgae, and macroalgae or as refugia for ecosystem resilience following environmental stress.

First Report of Endolithic Members of Rhodosorus marinus (Stylonematales, Rhodophyta) Growing Inside Rhodoliths Offshore Louisiana, Northwestern Gulf of Mexico

“…This study adds to our previous discovery that the interior of rhodoliths are marine biodiversity hotspots (Sauvage et al, 2016a;Krayesky-Self et al, 2017;Fredericq et al, 2019) for previously unknown endolithic stages in the life history of ecologically important and diverse microalgae encompassing phyla as diverse as dinoflagellates (i.e., Prorocentrum), haptophytes (i.e., Ochrosphaera), and, as noted herein, red algae (i.e., Rhodosorus) as well. The metabarcoding approach may reveal additional unknown biodiversity that can form the basis for species description through careful culturing efforts and anatomical observations, as was performed here.…”

“…S. Fredericq, depth 54-55 m, 26.viii.2012, LAF6573b) offshore Louisiana in the northwestern Gulf of Mexico aboard the R/V Pelican, a UNOLS research vessel operated out of LUMCON, Cocodrie, LA. Rhodoliths were retrieved using a Hourglass-design box dredge (Joyce and Williams, 1969) with minimum tows (usually 8 min or less) (Felder et al, 2014;Fredericq et al, 2014Fredericq et al, , 2019. Rhodoliths were initially stored on-site by location in containers filled with seawater collected in situ from the same depth and site of the sampled rhodoliths using the onboard CTD water-sampling rozette.…”

“…A 75-L closed microcosm tank, established from a subset of samples from Ewing Bank, was equipped with a SeaClone 100 protein skimmer (Instant Ocean, Blacksburg, VA, United States), water jet (MJ2000) and 600 lumen light (FugeRay Unibody). The protein skimmer provided filtration and a flow of 1,200 L/h (Krayesky-Self et al, 2017;Fredericq et al, 2019). The LED photosynthetically available radiation (PAR) in the microcosm was about 30 µmol photons m −2 s −1 , a measurement approximating in situ light PAR or irradiance levels measured with a LI-COR Biosciences (Lincoln, NE, United States) biospherical PAR sensor incorporated in a CTD (for conductivity, temperature, and depth) rosette and water sampler.…”

“…Whereas R. marinus and R. magnei typically grow epiphytically on the surface of seaweeds (Fresnel and Billard, 1995), we recently found a member of Rhodosorus growing inside (endolithically) calcified Lithothamnion (Hapalidiaceae, Hapalidiales) rhodoliths collected in mesophotic rhodolith beds in the northwestern Gulf of Mexico. Rhodoliths are free-living marine benthic spheroidal nodules predominantly accreted by crustose coralline red algae (CCA) precipitating CaCO 3 within their organic cell walls (Foster, 2001;Krayesky-Self et al, 2016Fredericq et al, 2019;Spalding et al, 2019). Two major rhodolith categories can be found in the northwestern Gulf of Mexico (NWGMx), i.e., biogenic and autogenic rhodoliths.…”

“…Autogenic rhodoliths are viewed as a specific type of nucleated rhodoliths (sensu Freiwald and Henrich, 1994) in which the core derives from calcium carbonate rubble as opposed to other materials. These two categories of rhodoliths co-inhabit the same rhodolith beds but the internal (endolithic) microbiome of each category may differ with regard to the diversity of taxa (biogenic: Krayesky-Self et al, 2017, andautogenic: Sauvage et al, 2016a;Fredericq et al, 2019).…”