Metabarcoding reveals unique microbial mat communities and evidence of biogeographic influence in low-oxygen, high-sulfur sinkholes and springs.

High-sulfur, low-oxygen environments formed by underwater sinkholes and springs create unique habitats populated by microbial mat communities. To explore the diversity and biogeography of these mats, samples were collected from three sites in Alpena, Michigan, one site in Monroe, Michigan, and one site in Palm Coast, Florida. Our study investigated previously undescribed eukaryotic diversity in these habitats and further explored their bacterial communities. Mat samples and water parameters were collected from sulfur spring sites during the spring, summer, and fall of 2022. Cyanobacteria and diatoms were cultured from mat subsamples to create a culture-based DNA reference library. Remaining mat samples were used for metabarcoding of the 16S and rbcL regions to explore bacterial and diatom diversity, respectively. Analyses of water chemistry, alpha diversity, and beta diversity articulated a range of high-sulfur, low-oxygen habitats, each with distinct microbial communities. Conductivity, pH, dissolved oxygen, temperature, sulfate, and chloride had significant influences on community composition but did not describe the differences between communities well. Chloride concentration had the strongest correlation with microbial community structure. Mantel tests revealed that biogeography contributed to differences between communities as well. Our results provide novel information on microbial mat composition and present evidence that both local conditions and biogeography influence these unique communities.

Unbiased analyses of ITS folding motifs in a taxonomically confusing lineage: Anagnostidinema visiae sp. nov. (cyanobacteria).

Cyanobacteria are diverse prokaryotic, photosynthetic organisms present in nearly every known ecosystem. Recent investigations around the world have recovered vast amounts of novel biodiversity in seldom sampled habitats. One phylogenetically significant character, the secondary folding structures of the 16S-23S ITS rDNA region, has allowed an unprecedented capacity to erect new species. However, two questions arise: Is this feature as informative as is proposed, and how do we best employ these features? Submerged sinkholes with oxygen-poor, sulfur-rich ground water in Lake Huron (USA) contain microbial mats dominated by both oxygenic and anoxygenic cyanobacteria. We sought to document some of this unique cyanobacterial diversity. Using culture-based investigations, we recovered 45 strains, of which 23 were analyzed employing 16S-23S rDNA sequences, ITS folding patterns, ecology, and morphology. With scant morphological discontinuities and nebulous 16S rDNA gene sequence divergence, ITS folding patterns were effective at articulating cryptic biodiversity. However, we would have missed these features had we not folded all the available motifs from the strains, including those with highly similar 16S rDNA gene sequences. If we had relied solely on morphological or 16S rDNA gene data, then we might well have missed the diversity of Anagnostidinema. Thus, in order to avoid conformation basis, which is potentially common when employing ITS structures, we advocate clustering strains based on ITS rDNA region patterns independently and comparing them back to 16S rDNA gene phylogenies. Using a total evidence approach, we erected a new taxon according to the International Code of Nomenclature for Algae, Fungi, and Plants: Anagnostidinema visiae.

Indications of symbiotic state influencing melanin-synthesis immune response in the facultative coral Astrangia poculata.

Increased ocean warming is causing detrimental impacts to tropical corals worldwide. Compounding the effects of heat stress, incidences of tropical coral disease have risen concurrently. While tropical coral responses to these impacts are well studied, temperate coral responses remain largely unknown. The present study focused on the immune response of the temperate coral Astrangia poculata to increased temperature and disease. Symbiotic and aposymbiotic A. poculata were collected from Narragansett Bay, Rhode Island (USA) in summer and winter seasons and exposed to control (18°C) versus elevated temperatures (26°C) in the presence of an immune stimulant (i.e. lipopolysaccharide) for a 12 h period. Prophenoloxidase (PPO) and melanin concentrations from the melanin-synthesis pathway were assessed via spectrophotometry to examine immune responses. While PPO measurements were higher on average in symbiotic corals compared with aposymbiotic corals, temperature and season did not significantly affect this metric. Melanin was significantly higher in symbiotic compared to aposymbiotic corals, implying that symbiotic state may be important for melanin-synthesis response. Conversely, melanin as an immune response may be of less importance in aposymbiotic A. poculata due to the potential capacity of other immune responses in this species. In addition, differences in resource allocation to immune investment as a result of symbiosis is plausible given melanin production observed within the present study. However, thermal stressors may reduce the overall influence of symbiosis on melanin production. Future studies should build upon these results to further understand the entirety of innate immunity responses in temperate coral species.

Extensive chloroplast genome rearrangement amongst three closely related Halamphora spp. (Bacillariophyceae), and evidence for rapid evolution as compared to land plants.

Diatoms are the most diverse lineage of algae, but the diversity of their chloroplast genomes, particularly within a genus, has not been well documented. Herein, we present three chloroplast genomes from the genus Halamphora (H. americana, H. calidilacuna, and H. coffeaeformis), the first pennate diatom genus to be represented by more than one species. Halamphora chloroplast genomes ranged in size from ~120 to 150 kb, representing a 24% size difference within the genus. Differences in genome size were due to changes in the length of the inverted repeat region, length of intergenic regions, and the variable presence of ORFs that appear to encode as-yet-undescribed proteins. All three species shared a set of 161 core features but differed in the presence of two genes, serC and tyrC of foreign and unknown origin, respectively. A comparison of these data to three previously published chloroplast genomes in the non-pennate genus Cyclotella (Thalassiosirales) revealed that Halamphora has undergone extensive chloroplast genome rearrangement compared to other genera, as well as containing variation within the genus. Finally, a comparison of Halamphora chloroplast genomes to those of land plants indicates diatom chloroplast genomes within this genus may be evolving at least ~4-7 times faster than those of land plants. Studies such as these provide deeper insights into diatom chloroplast evolution and important genetic resources for future analyses.

Comparative analysis of the mitochondrial genomes of six newly sequenced diatoms reveals group II introns in the barcoding region of cox1.

Diatoms are the most diverse lineage of algae and at the base of most aquatic food webs, but only 11 of their mitochondrial genomes have been described. Herein, we present the mitochondrial genomes of six diatom species, including: Melosira undulata, Nitzschia alba, Surirella sp., Entomoneis sp., Halamphora coffeaeformis, and Halamphora calidilacuna. Comparison of these six genomes to the 11 currently published diatom mitochondrial genomes revealed a novel ubiquitous feature block consisting of tatC-orf157-rps11. The presence of intronic retrotransposable elements in the barcoding region of cox1 in the Halamphora genomes may explain historic difficulty (especially PCR) with cox1 as a universal barcode for diatoms. Our analysis suggests that high rates of variability in number and position of introns, in many commonly used coding sequences, prevent these from being universally viable as barcodes for diatoms. Therefore, we suggest researchers examine the chloroplast and/or nuclear genomes for universal barcoding markers.

Effects of Temperature and Photorepair Radiation on a Marine Ciliate Exposed to UVB Radiation.

The influences of intensity and repeated exposure to ultraviolet-B radiation (UVB), photoreactivating repair radiation (PRR), and temperature on the scuticociliate Parauronema acutum were explored under laboratory conditions. Population growth was negatively affected after exposure to the equivalent of one sunny summer day of ambient UVB, especially in the absence of PRR. Repeated daily exposure to UVB severely compromised ciliate survival. UVB-exposed treatments without PRR recovered slower and reached lower final abundances than treatments receiving PRR. Reducing the daily UVB exposure approximately 25% improved ciliate recovery after exposure. In the single exposure treatments, temperature effects were not consistent, except that growth was slowest for control and treatments at the lowest temperature (15 °C). These data suggest that dark repair and/or photoprotection are present in P. acutum, but photoenzymatic repair was the more effective mechanism in reversing UVB damage. Repeated exposure treatments without PRR had zero or declining growth at all temperatures (15, 20 and 25 °C), as did those with PRR at 15 °C. Significant temperature/dose differences were identified in the repeated exposure treatments; ciliates subjected to the higher UVB intensity with PRR survived only at 25 °C, while ciliate populations under reduced UVB increased at 20 and 25 °C.

A comparison of morphological and molecular-based surveys to estimate the species richness of Chaetoceros and Thalassiosira (bacillariophyta), in the Bay of Fundy.

The goal of this study was to compare the ability of morphology and molecular-based surveys to estimate species richness for two species-rich diatom genera, Chaetoceros Ehrenb. and Thalassiosira Cleve, in the Bay of Fundy. Phytoplankton tows were collected from two sites at intervals over two years and subsampled for morphology-based surveys (2010, 2011), a culture-based DNA reference library (DRL; 2010), and a molecular-based survey (2011). The DRL and molecular-based survey utilized the 3' end of the RUBISCO large subunit (rbcL-3P) to identify genetic species groups (based on 0.1% divergence in rbcL-3P), which were subsequently identified morphologically to allow comparisons to the morphology-based survey. Comparisons were compiled for the year (2011) by site (n = 2) and by season (n = 3). Of the 34 taxa included in the comparisons, 50% of taxa were common to both methods, 35% were unique to the molecular-based survey, and 12% were unique to the morphology-based survey, while the remaining 3% of taxa were unidentified genetic species groups. The morphology-based survey excelled at identifying rare taxa in individual tow subsamples, which were occasionally missed with the molecular approach used here, while the molecular methods (the DRL and molecular-based survey), uncovered nine cryptic species pairs and four previously overlooked species. The last mentioned were typically difficult to identify and were generically assigned to Thalassiosira spp. during the morphology-based survey. Therefore, for now we suggest a combined approach encompassing routine morphology-based surveys accompanied by periodic molecular-based surveys to monitor for cryptic and difficult to identify taxa. As sequencing technologies improve, molecular-based surveys should become routine, leading to a more accurate representation of species composition and richness in monitoring programs.

Barcoding diatoms: exploring alternatives to COI-5P.

Diatoms are a diverse lineage with species that can be difficult to identify or cryptic, but DNA barcoding, a molecular technique, can assist identification and facilitate studies of speciation and biogeography. The most common region used for DNA barcoding, COI-5P, can distinguish diatom species, but has not displayed universality (i.e., successful PCR amplification from diverse taxa). Therefore, we have assessed the following alternative markers: ∼1400bp of rbcL; 748bp at the 3' end of rbcL (rbcL-3P); LSU D2/D3 and UPA. Sellaphora isolates were used to determine each marker's ability to discriminate among closely related species and culture collection material was utilized to explore further marker universality. All of the alternative markers investigated have greater universality than COI-5P. Both full and partial (3P) rbcL regions had the power to discriminate between all species, but rbcL-3P can be sequenced more easily. LSU D2/D3 could distinguish between all but the most closely related species (96%), whereas UPA only distinguished 20% of species. Our observations suggest that rbcL-3P should be used as the primary marker for diatom barcoding, while LSU D2/D3 should be sequenced as a secondary marker to facilitate environmental surveys.