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.

Interspecific systematics in the red algal order Sporolithales remains problematic. To re-evaluate its species, DNA analyses were performed on historical type material and recently collected specimens assigned to the two genera Sporolithon and Heydrichia. Partial rbcL sequences from the lectotype specimens of Sporolithon ptychoides (the generitype species) and Sporolithon molle, both from El Tor, Egypt, are exact matches to field-collected topotype specimens. Sporolithon crassum and Sporolithon erythraeum also have the same type locality; material of the former appears to no longer exist, and we were unable to PCR amplify DNA from the latter. A new species, Sporolithon eltorensis, is described from the same type locality. We have not found any morpho-anatomical characters that distinguish these three species. No sequenced specimens reported as S. ptychoides from other parts of the world represent this species, and likely reports of S. ptychoides and S. molle based on morpho-anatomy are incorrect. A partial rbcL sequence from the holotype of Sporolithon dimotum indicates it is not a synonym of S. ptychoides, and data from the holotype of S. episporum confirm its specific recognition. DNA sequences from topotype material of Heydrichia woelkerlingii, the generitype species, and isotype material of Heydrichia cerasina confirm that these are distinct species; the taxon reported to be H. woelkerlingii from New Zealand is likely an undescribed species. Type specimens of all other Sporolithon and Heydrichia species need to be sequenced to confirm that they are distinct species; morpho-anatomical studies have proved inadequate for this task.

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Hard bank rhodolith beds at 45-80 m depth offshore Louisiana in the Northwestern Gulf of Mexico harbor a diverse community of non-geniculate coralline algae spanning multiple lineages including both rhodolith-forming (biogenic) taxa and others encrusting autogenic rhodoliths. Identifying these members of the Corallinales to the correct genus and species is an ongoing process because many available names need to be validated by comparison to historical type specimens. A phylogenetic analysis of concatenated plastid (psbA), nuclear (LSU rDNA), and mitochondrial (COI) DNA sequences of non-geniculate corallines belonging to the subfamily Lithophylloideae (Corallinaceae), including newly generated sequences from recently collected specimens dredged at Ewing and Sackett Banks following the April 2010 Deepwater Horizon oil spill, reveals at least two distinct species of Lithophyllum sensu lato for the region. Scanning Electron Microscopy confirmed the presence of vegetative characters congruent with those for both Lithophyllum and Titanoderma. Lithophyllum is a newly reported genus for the northern Gulf of Mexico. The generic boundaries within the Lithophylloideae are addressed in light of possible evolutionary progenetic heterochrony that may have occurred within this subfamily.

In the Northwestern Gulf of Mexico (NWGMx), subtidal rhodolith beds offshore Louisiana at 45-80 m depth harbor a diverse community of uncharacterized non-geniculate coralline algae including both biogenic and autogenic rhodoliths and other encrusting taxa. Identifying specimens to their correct genus and species is an ongoing process because many available names remain to be validated by comparison to type specimens. Here, comparative DNA sequencing (psbA, UPA, and COI) and scanning electron microscopy (SEM) are used to assess the molecular and morphological diversity of the rhodolithforming specimens belonging to the generic concept of Lithothamnion. Phylogenetic and species delimitation analyses of the newly generated sequences from recently dredged specimens at Ewing and Sackett Banks offshore Louisiana reveal the presence of at least six species of Lithothamnion, whose generic placement is confirmed by SEM images of features considered characteristic for the genus. More broadly, our analyses indicate at least eight Lithothamnion species are found in the Gulf of Mexico. Phylogenetic analyses of single (psbA and COI) and concatenated markers (psbA, COI and UPA) show that Lithothamnion is polyphyletic.