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Background: In spite of their ecological importance as primary producers and microbioeroders of marine calcium carbonate (CaCO 3 ) substrata, endolithic phototrophs spanning both prokaryotic (the cyanobacteria) and eukaryotic algae lack established molecular resources for their facilitated survey with high throughput sequencing. Here, the development of a metabarcoding framework for the elongation factor EF-Ttu (tufA) was tested on four Illuminasequenced marine CaCO 3 microfloras for the characterization of their endolithic phototrophs, especially the abundant bioeroding Ostreobium spp. (Ulvophyceae). The framework consists of novel tufA degenerate primers and a comprehensive database enabling Operational Taxonomic Unit (OTU) identification at multiple taxonomic ranks with percent identity thresholds determined herein. Results:The newly established tufA database comprises 4057 non-redundant sequences (from 1339 eukaryotic and prokaryotic phototrophs, and 2718 prokaryotic heterotrophs) including 27 classes in 10 phyla of phototrophic diversity summarized from data mining on GenBank ® , our barcoding of >150 clones produced from coral reef microfloras, and >300 eukaryotic phototrophs (>230 Ulvophyceae including >100 'Ostreobium' spp., and >70 Florideophyceae, Phaeophyceae and miscellaneous taxa). Illumina metabarcoding with the newly designed primers resulted in 802 robust OTUs including 618 phototrophs and 184 heterotrophs (77 and 23 % of OTUs, respectively). Phototrophic OTUs belonged to 14 classes of phototrophs found in seven phyla, and represented ~98 % of all reads. The phylogenetic profiles of coral reef microfloras showed few OTUs in large abundance (proportion of reads) for the Chlorophyta (Ulvophyceae, i.e. Ostreobium and Phaeophila), the Rhodophyta (Florideophyceae) and Haptophyta (Coccolithophyceae), and a large diversity (richness) of OTUs in lower abundance for the Cyanophyta (Cyanophyceae) and the Ochrophyta (the diatoms, 'Bacillariophyta'). The bioerosive 'Ostreobium' spp. represented four families in a large clade of subordinal divergence, i.e. the Ostreobidineae, and a fifth, phylogenetically remote family in the suborder Halimedineae (provisionally assigned as the 'Pseudostreobiaceae'). Together they harbor 85-95 delimited cryptic species of endolithic microsiphons. Conclusions:The novel degenerate primers allowed for amplification of endolithic phototrophs across a wide phylogenetic breadth as well as their recovery in very large proportions of reads (overall 98 %) and diversity (overall 77 % of OTUs). The established companion tufA database and determined identity thresholds allow for OTU identification at multiple taxonomic ranks to facilitate the monitoring of phototrophic assemblages via metabarcoding, especially endolithic communities rich in bioeroding Ulvophyceae, such as those harboring 'Ostreobium' spp., Phaeophila spp. and associated algal diversity.

The red alga Gracilaria salicornia (c. Agardh) E. Y. Dawson was introduced intentionally to two reefs on O'ahu, Hawai'i, in the 1970s for experimental aquaculture for the agar industry. Some 30 yr later, this species has spread from the initial sites of introduction and is now competing with native marine flora and fauna. The goals of this study were to quantify various aspects of G. salicornia ecology in Hawai'i in an effort to develop control or eradication tools. Experimental plots were established to determine cover and biomass of G. salicornia per square meter and to determine the amount of time and person hours needed to remove G. salicornia from these plots. Substantial amounts of G. salicornia become dislodged from the reef during large wave events and periodically become deposited onto the beach in front of the WaikIkI Aquarium. Algal beach wash biomass was quantified and positive relationships were established between swell height and the amount of algae that washed up onto the beach in this location. We then quantified the ability of G. salicornia vegetative fragments to regrow after desiccation to determine if algal biomass stranded on shore survives the tidal cycle until being washed back out on the reef at high tide. Gracilaria salicornia was remarkably resistant to temperature, salinity, and chemical treatments examined as possible in situ control options. Herbivore preference tests showed that a native Gracilaria species is consumed far more frequently than the alien congener. Finally, large-scale community volunteer efforts were organized to remove drifting G. salicornia fragments from the reef area in front of the WaikIkI Aquarium. Over 20,000 kg of alien algal fragments were removed from this location in five 4-hr cleanup events. However, based on G. salicornia growth rates, ability to fragment, physical tolerance, and low herbivory, it is clear that a large-scale dedicated effort will be needed to control this invasive species on WaikIkI's reefs. THE LARGE-SCALE introduction of nonindigenous species is among the greatest threats to the homogenization of the world's biota and subsequent reduction in global biodiversity (Carlton and Geller 1993, Maragos et al. 1996, Ruiz et al. 1999

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.

BackgroundThe Hawaiian red algal flora is diverse, isolated, and well studied from a morphological and anatomical perspective, making it an excellent candidate for assessment using a combination of traditional taxonomic and molecular approaches. Acquiring and making these biodiversity data freely available in a timely manner ensures that other researchers can incorporate these baseline findings into phylogeographic studies of Hawaiian red algae or red algae found in other locations.ResultsA total of 1,946 accessions are represented in the collections from 305 different geographical locations in the Hawaiian archipelago. These accessions represent 24 orders, 49 families, 152 genera and 252 species/subspecific taxa of red algae. One order of red algae (the Rhodachlyales) was recognized in Hawaii for the first time and 196 new island distributional records were determined from the survey collections. One family and four genera are reported for the first time from Hawaii, and multiple species descriptions are in progress for newly discovered taxa. A total of 2,418 sequences were generated for Hawaiian red algae in the course of this study - 915 for the nuclear LSU marker, 864 for the plastidial UPA marker, and 639 for the mitochondrial COI marker. These baseline molecular data are presented as neighbor-joining trees to illustrate degrees of divergence within and among taxa. The LSU marker was typically most conserved, followed by UPA and COI. Phylogenetic analysis of a set of concatenated LSU, UPA and COI sequences recovered a tree that broadly resembled the current understanding of florideophyte red algal relationships, but bootstrap support was largely absent above the ordinal level. Phylogeographic trends are reported here for some common taxa within the Hawaiian Islands and include examples of those with, as well as without, intraspecific variation.ConclusionsThe UPA and COI markers were determined to be the most useful of the three and are recommended for inclusion in future algal biodiversity surveys. Molecular data for the survey provide the most extensive assessment of Hawaiian red algal diversity and, in combination with the morphological/anatomical and distributional data collected as part of the project, provide a solid baseline data set for future studies of the flora. The data are freely available via the Hawaiian Algal Database (HADB), which was designed and constructed to accommodate the results of the project. We present the first DNA sequence reference collection for a tropical Pacific seaweed flora, whose value extends beyond Hawaii since many Hawaiian taxa are shared with other tropical areas.

The genus Pseudochlorodesmis (Bryopsidales) is composed of diminutive siphons of extreme morphological simplicity. The discovery of Pseudochlorodesmis-like juveniles in more complex Bryopsidales (e.g., the Halimeda microthallus stage) jeopardized the recognition of this genus. Confronted with this uncertainty, taxonomists transferred many simple siphons into a new genus, Siphonogramen. In this study, we used a multimarker approach to clarify the phylogenetic and taxonomic affinities of the Pseudochlorodesmis-Siphonogramen (PS) complex within the more morphologically complex bryopsidalean taxa. Our analyses reveal a new layer of diversity largely distinct from the lineages containing the structurally complex genera. The PS complex shows profound cryptic diversity exceeding the family level. We discuss a potential link between thallus complexity and the prevalence and profundity of cryptic diversity. For taxonomic simplicity and as a first step toward clarifying the taxonomy of these simple siphons, we propose to maintain Pseudochlorodesmis as a form genus and subsume Siphonogramen and Botryodesmis therein.

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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