Fine-tuning RNA extraction protocols for polysaccharide-rich freshwater red algae (Rhodophyta).

Freshwater red algae are important primary producers, widely distributed, contributing significantly to nutrient cycling in aquatic ecosystems. Recent studies have focused on identifying the effect of different environmental conditions such as light on the gene expression in photosynthesis pathways. However, obtaining the necessary RNA quantity and quality for sequencing from these algae has been challenging. Although RNA extractions have been optimized for model organisms, RNA extraction for non-model organisms, such as gelatinous (polysaccharide-rich) red algae, requires considerable troubleshooting. The common freshwater red alga, Batrachospermum gelatinosum , was used to test protocols. The extraction efficiency of various sample disruption methods in combination with seven RNA extraction kits was compared. Using a 2-minute disruption procedure with a modification of TRIzol ™ Plus RNA Purification Kit (PureLink ™ RNA Mini Kit + Trizol ™ ) protocol resulted in significantly higher RIN scores (p < 0.05) and high RNA concentration, compared to other methods. The fine-tuned protocol yielded quality RNA (RIN>7) in high concentrations for subsequent sequencing.

The eco-evolutionary importance of reproductive system variation in the macroalgae: Freshwater reds as a case study.

The relative frequency of sexual versus asexual reproduction governs the distribution of genetic diversity within and among populations. Most studies on the consequences of reproductive variation focus on the mating system (i.e., selfing vs. outcrossing) of diploid-dominant taxa (e.g., angiosperms), often ignoring asexual reproduction. Although reproductive systems are hypothesized to be correlated with life-cycle types, variation in the relative rates of sexual and asexual reproduction remains poorly characterized across eukaryotes. This is particularly true among the three major lineages of macroalgae (green, brown, and red). The Rhodophyta are particularly interesting, as many taxa have complex haploid-diploid life cycles that influence genetic structure. Though most marine reds have separate sexes, we show that freshwater red macroalgae exhibit patterns of switching between monoicy and dioicy in sister taxa that rival those recently shown in brown macroalgae and in angiosperms. We advocate for the investigation of reproductive system evolution using freshwater reds, as this will expand the life-cycle types for which these data exist, enabling comparative analyses broadly across eukaryotes. Unlike their marine cousins, species in the Batrachospermales have macroscopic gametophytes attached to filamentous, often microscopic sporophytes. While asexual reproduction through monospores may occur in all freshwater reds, the Compsopogonales are thought to be exclusively asexual. Understanding the evolutionary consequences of selfing and asexual reproduction will aid in our understanding of the evolutionary ecology of all algae and of eukaryotic evolution generally.

Paludicola gen. nov. and Revision of the Species Formerly in Batrachospermum Section Turfosa (Batrachospermales, Rhodophyta).

Since the first phylogenetic study of the order Batrachospermales, Batrachospermum was shown to be paraphyletic. Subsequently, sections of the genus have been methodically investigated using DNA sequences and morphology in order to propose new genera and delineate species. Batrachospermum section Turfosa is the last section with multiple species yet to be examined. New sequence data of specimens from Europe and the United States were combined with the sparse sequence data already available. Phylogenetic analyses using rbcL and COI-5P sequences showed this section to be a well-supported clade, distinct from Batrachospermum section Batrachospermum and its segregate genera. Section Turfosa is raised to the generic rank as Paludicola gen. nov. Substantial genetic variation within the genus was discovered and 12 species are recognized based on DNA sequence data as well as morphological characters and geographic distribution. The following morphological characters were applied to distinguish species: branching pattern (pseudodichotomous or irregular), whorl size (reduced or well developed), primary fascicles (curved or straight), spermatangia origin (primary or secondary fascicles), and carposporophyte arrangement (loose or dense). Previously published species were transferred to the new genus: P. turfosa, P. keratophyta, P. orthosticha, P. phangiae, and P. periploca. Seven new species are proposed as follows: P. groenbladii from Europe; P. communis, P. johnhallii, and P. leafensis from North America; and P. aquanigra, P. diamantinensis, and P. turfosiformis from Brazil. In addition, three unsequenced species in the section, P. bakarensis, P. gombakensis, and P. tapirensis, were transferred to the new genus.

Relative expression analysis of light-harvesting genes in the freshwater alga Lympha mucosa (Batrachospermales, Rhodophyta).

Members of the freshwater red algal order Batrachospermales are often described as shade-adapted. Nevertheless, recent ecophysiological studies have demonstrated species-level differences in acclimation to a range of irradiances. Lympha mucosa occurs in open and shaded portions of temperate streams and is abundant during summer months, suggesting it tolerates high and low irradiances. Specimens of L. mucosa were collected from open (sun-acclimated) or shaded (shade-acclimated) sites and exposed to low (<20 µmol photons · m-2 · s-1 ) or high (220 µmol photon · m-2 · s-1 ) light for 72 h to examine mechanisms of photoacclimation at the transcriptional level. High-throughput sequence data were used to design specific primers for genes involved with light harvesting and these were quantified with qPCR. The greatest significant difference in transcript abundances was observed in the psaA gene (Photosystem I P700 apoprotein), and site-type had an effect on these responses. Shade-acclimated thalli were 22-fold down-regulated at high light, whereas sun-acclimated thalli were only 5-fold down-regulated. Another gene involved with Photosystem I (petF ferredoxin) was down-regulated at high light, but only individuals from the shaded site were significantly different (4-fold). In thalli from both sites, cpeA (Phycoerythrin alpha chain) was down-regulated at high light. Although not statistically significant, patterns consistent with previous physiological and transcriptomic studies were uncovered, namely the inverse response of transcriptional activity in genes that encode phycobiliproteins. In support of previous ecophysiological studies of freshwater red algae, these data indicate significant transcriptional changes involving Photosystem I and phycobiliprotein synthesis are required to tolerate and grow at various irradiances.

Stream Algal Biofilm Community Diversity Along An Acid Mine Drainage Recovery Gradient Using Multimarker Metabarcoding.

In southeastern Ohio, active remediation of streams affected by Acid Mine Drainage (AMD) has proven to be successful for some streams, while others have not recovered based on macroinvertebrate assessment. In this study, biofilms were collected from three Moderately Impaired, three Recovered, and two Unimpaired streams. The biodiversity was characterized by metabarcoding using two universal barcode markers (16S and 18S) along with two algal specific markers (UPA and rbcL) and high-throughput amplicon sequencing. For each marker, the ordination of Bray-Curtis Index calculated from the total Amplicon Sequence Variants (ASVs) present in each stream showed the Unimpaired and Recovered streams clustered, while Moderately Impaired streams were more distant. Focusing on the algal ASVs, the Shannon index for the rbcL, and UPA markers showed significantly lower alpha diversity in Moderately Impaired streams compared to Unimpaired streams, but the Recovered streams were not significantly different from the other two stream categories. The two universal markers together captured all algal phyla providing an outline of the diversity, but the two algal specific markers produced a greater number of ASVs and taxonomic depth for algal taxa. Further examination of the UPA marker revealed a drastic decrease in relative abundance of diatoms in Moderately Impaired streams compared to Recovered and Unimpaired streams. Likewise, diatom genera identified in the rbcL data and indicative of stream water quality showed marked differences in relative abundance among stream categories. Although all markers were useful, the algal-specific UPA and rbcL contributed more insights into algal community differences among stream categories.

Seasonality of total fatty acid profiles in acid mine drainage impaired streams.

Pre-regulation coal mining and subsequent acid mine drainage (AMD) have drastically altered stream quality in the Appalachian region of the USA. Streams impaired by AMD often times demonstrate lowered pH, increases in specific conductance, and increase in dissolved metal concentrations. These changes in the chemical environment are reflected in the biotic community with drastic reductions in diversity and biomass. Recently, there has been an increase in applying traditional measures of food quality to understand how the biofilm community is altered by environmental condition and use for stream quality monitoring. The purpose of this study was two-fold: (1) to use fatty acid profiles to distinguish between biofilm communities in AMD impaired and unimpaired streams and (2) to determine the consistency of biofilm fatty acid profiles throughout the summer sampling period. Impaired streams showed significantly lower pH and increased specific conductance. Biofilm samples from the AMD impaired streams had lower fatty acid content with a decreased proportion of polyunsaturated fatty acids. Fatty acid profiles easily and rapidly separated biofilm communities into their respective categories, either as being impaired by AMD or unimpaired by AMD, using multivariate statistical approaches. Fatty acid profiles were similar within stream type throughout the summer sampling season, and the profiles were correlated to pH and specific conductance. The results of this study suggest that fatty acid profiles can rapidly and accurately categorize the biofilm community responses to environmental impairment.

Three gene phylogeny of the Thoreales (Rhodophyta) reveals high species diversity.

The freshwater red algal order Thoreales has triphasic life history composed of a diminutive diploid "Chantransia" stage, a distinctive macroscopic gametophyte with multi-axial growth and carposporophytes that develop on the gametophyte thallus. This order is comprised of two genera, Thorea and Nemalionopsis. Thorea has been widely reported with numerous species, whereas Nemalionopsis has been more rarely observed with only a few species described. DNA sequences from three loci (rbcL, cox1, and LSU) were used to examine the phylogenetic affinity of specimens collected from geographically distant locations including North America, South America, Europe, Pacific Islands, Southeast Asia, China, and India. Sixteen species of Thorea and two species of Nemalionopsis were recognized. Morphological observations confirmed the distinctness of the two genera and also provided some characters to distinguish species. However, many of the collections were in "Chantransia" stage rather than gametophyte stage, meaning that key diagnostic morphological characters were unavailable. Three new species are proposed primarily based on the DNA sequence data generated in this study, Thorea kokosinga-pueschelii, T. mauitukitukii, and T. quisqueyana. In addition to these newly described species, one DNA sequence from GenBank was not closely associated with other Thorea clades and may represent further diversity in the genus. Two species in Nemalionopsis are recognized, N. shawii and N. parkeri nom. et stat. nov. Thorea harbors more diversity than had been recognized by morphological data alone. Distribution data indicated that Nemalionopsis is common in the Pacific region, whereas Thorea is more globally distributed. Most species of Thorea have a regional distribution, but Thorea hispida appears to be cosmopolitan.

Ottia meiospora (Ottiaceae, Rhodophyta), a new genus and family endophytic within the thallus of Nothocladus (Batrachospermales, Rhodophyta).

A new genus, Ottia, and family, Ottiaceae, are proposed within the Acrochaetiales to accommodate the uniseriate red algal endophyte of batrachspermalean taxa previously named Balbiania meiospora. Prior to this study, Balbiania investiens was transferred to its own family and order (Balbianiales) based on comparative DNA sequence data and a distinctive reproductive morphology. However, the second species described in this genus, B. meiospora, continued to be treated as a species of Audouinella (A. meiospora) pending further investigation. Phylogenetic analyses of sequence data confirmed only a distant relationship between the two endophytes, and a closer alliance of B. meiospora to Acrochaetiales. The data also showed that Ottia meiospora was the deepest diverging lineage in the Acrochaetiales, sister to all of the currently recognized genera and families. In this study, we review the classification of what we now call O. meiospora - reported from Australia, New Zealand and Brazil - based on sequence and morphological data. Morphological observations provided little clarity around the reproductive morphology or the life cycle of this endophyte of Nothocladus s. lat. found commonly in mainland Australia but, to date, less so in New Zealand.

Complete mitochondrial genome of the freshwater red alga Lympha mucosa (Rhodophyta).

We present the complete mitochondrial genome of a newly described freshwater red alga Lympha mucosa. The genome was sequenced using the Illumina MiSeq platform. The circular mitochondrial genome is 25,191 bp, contains 46 genes (24 CDS, 20 tRNA, and 2 rRNA), and has an overall GC content of 27.5%. Phylogenetic analyses of the cox1 gene show the placement of Lympha mucosa within the strictly freshwater order Batrachospermales. The four mitochondrial genomes within the subclass Nemaliophycidae sequenced to date are highly conserved in terms of genome size, gene content, and gene synteny.

Nocturama gen. nov., Nothocladus s. lat. and other taxonomic novelties resulting from the further resolution of paraphyly in Australasian members of Batrachospermum (Batrachospermales, Rhodophyta).

The informal "Australasica Group" was established in 2009 to include several Australasian endemic Batrachospermum species, a few species of the cosmopolitan Batrachospermum section Setacea, and the South American endemic Petrohua bernabei. Although useful for communication purposes, no formal taxonomic designation was proposed due to weakly supported basal nodes. The present research took a two-pronged approach of adding more taxa (29 additional specimens) as well as more sequence data (LSU, cox1, psaA, and psbA markers added to rbcL data) to provide better resolution. The resulting tree showed improved statistical support values (Bayesian posterior probability and maximum likelihood bootstrap) for most nodes providing a framework for taxonomic revision. Based on our well-resolved phylogeny, a new genus, Nocturama, is proposed for a clade of Batrachospermum antipodites specimens. The circumscription of Nothocladus is expanded to include Batrachospermum section Setacea and four additional sections composed of at least 10 species, mostly from Australia and New Zealand. One new species added to the data set, N. diatyches, did not form a clade with the other species of section Setaceus, where it was classified previously, rendering that section paraphyletic. To resolve this, N. diatyches and the morphologically similar species N. latericius are included with N. theaquus, in the new section Theaquus within Nothocladus s. lat. A specimen from Australia unaligned to these clades was sister to the Australia-New Zealand genus Psilosiphon and the cosmopolitan B. cayennense, but lacked statistical support. This specimen has the gross morphology of Batrachospermum s. lat. and is here provisionally assigned to that genus, as B. serendipidum sp. nov.

Using fatty acids to fingerprint biofilm communities: a means to quickly and accurately assess stream quality.

The assessment of lotic ecosystem quality plays an essential role to help determine the extent of environmental stress and the effectiveness of restoration activities. Methods that incorporate biological properties are considered ideal because they provide direct assessment of the end goal of a vigorous biological community. Our primary objective was to use biofilm lipids to develop an accurate biomonitoring tool that requires little expertise and time to facilitate assessment. A model was created of fatty acid biomarkers most associated with predetermined stream quality classification, exceptional warm water habitat (EWH), warm water habitat (WWH), and limited resource (LR-AMD), and validated along a gradient of known stream qualities. The fatty acid fingerprint of the biofilm community was statistically different (P = 0.03) and was generally unique to recognized stream quality. One striking difference was essential fatty acids (DHA, EPA, and ARA) were absent from LR-AMD and only recovered from WWH and EWH, 45 % more in EWH than WWH. Independently testing the model along a stream quality gradient, this model correctly categorized six of the seven sites, with no match due to low sample biomass. These results provide compelling evidence that biofilm fatty acid analysis can be a sensitive, accurate, and cost-effective biomonitoring tool. We conceive of future studies expanding this research to more in-depth studies of remediation efforts, determining the applicable geographic area for the method and the addition of multiple stressors with the possibility of distinguishing among stressors.

Multigene phylogeny of the red algal subclass Nemaliophycidae.

The red algae (Rhodophyta) are a lineage of primary endosymbionts whose ancestors represent some of the first photosynthetic eukaryotes on the planet. They primarily inhabit marine ecosystems, with only ∼5% of species found in freshwater systems. The subclass Nemaliophycidae is very diverse in ecological and life history features and therefore a useful model to study these traits, but the phylogenetic relationships among the orders are, for the most part, poorly resolved. To elucidate the phylogeny of the Nemaliophycidae, we constructed a nine-gene dataset comprised of nuclear, plastid, and mitochondrial markers for 67 red algal specimens. The resulting maximum likelihood (ML) phylogeny confirmed the monophyly of all orders. The sister relationship of the Acrochaetiales and Palmariales received high support and the relationship of the Balliales with Balbianiales and Entwisleiales with Colaconematales was moderately supported. The Nemaliales, Entwisleiales, Colaconematales, Palmariales and Acrochaetiales formed a highly supported clade. Unfortunately, all other relationships among the orders had low bootstrap support. Although the ML analysis did not resolve many of the relationships, further analyses suggested that a resolution is possible. A Phycas analysis supported a dichotomously branching tree and Bayesian analysis showed a similar topology with all relationships highly supported. Simulations extrapolating the number of nucleotide characters beyond the current size of the dataset suggested that most nodes in the phylogeny would be resolved if more data become available. Phylogenomic approaches will be necessary to provide a well-supported phylogeny of this subclass with all relationships resolved such that the evolution of freshwater species from marine ancestors as well as reproductive traits can be explored.

Roles of benthic algae in the structure, function, and assessment of stream ecosystems affected by acid mine drainage.

Tens of thousands of stream kilometers worldwide are degraded by a legacy of acid loads, high metal concentrations, and altered habitat caused by acid mine drainage (AMD) from abandoned underground and surface mines. As the primary production base in streams, the condition of algal-dominated periphyton communities is particularly important to nutrient cycling, energy flow, and higher trophic levels. Here, we synthesize current knowledge regarding how AMD-associated stressors affect (i) algal communities and their use as ecological indicators, (ii) their functional roles in stream ecosystems, and (iii) how these findings inform management decisions and evaluation of restoration effectiveness. A growing body of research has found ecosystem simplification caused by AMD stressors. Species diversity declines, productivity decreases, and less efficient nutrient uptake and retention occur as AMD severity increases. New monitoring approaches, indices of biological condition, and attributes of algal community structure and function effectively assess AMD severity and effectiveness of management practices. Measures of ecosystem processes, such as nutrient uptake rates, extracellular enzyme activities, and metabolism, are increasingly being used as assessment tools, but remain in their infancy relative to traditional community structure-based approaches. The continued development, testing, and implementation of functional measures and their use alongside community structure metrics will further advance assessments, inform management decisions, and foster progress toward restoration goals. Algal assessments will have important roles in making progress toward improving and sustaining the water quality, ecological condition, and ecosystem services of streams in regions affected by the legacy of unregulated coal mining.

Molecular and morphological evidence for Sheathia gen. nov. (Batrachospermales, Rhodophyta) and three new species.

The freshwater red algal genus Batrachospermum has been shown to be paraphyletic since the first molecular studies of the Batrachospermales. Previous research, along with this study, provides strong support for the clade Batrachospermum section Helminthoidea. This study has found that heterocortication, the presence of both cylindrical and bulbous cells on the main axis, is an underlying synapomorphy of this clade. Based on support from DNA sequences of the rbcL gene, the COI barcode region and the rDNA ITS 1 and 2, along with morphological studies, the new genus Sheathia is proposed. Seven heterocorticate species were recognized from the molecular clades. Sheathia boryana and S. exigua sp. nov. appear to be restricted to Europe, whereas S. confusa occurs in Europe and New Zealand. Sheathia involuta is widespread in the USA and reported for the first time from Europe. Sheathia americana sp. nov., has been collected in the USA and Canada, and S. heterocortica and S. grandis sp. nov. have been collected only in the USA. Sheathia confusa and S. grandis can be distinguished based on morphological characters, whereas DNA sequence data are required to conclusively distinguish the other species. Sheathia fluitans and S. carpoinvolucra also are placed within this genus based on the presence of heterocortication. These data also hint at greater diversity among non-heterocorticate Sheathia than is recognized by the single species name S. arcuata.

MOLECULAR PHYLOGENY OF THE GENUS KUMANOA (BATRACHOSPERMALES, RHODOPHYTA)(1).

Species belonging to the newly established genus Kumanoa were sampled from locations worldwide. DNA sequence data from the rbcL gene, cox1 barcode region, and universal plastid amplicon (UPA) were collected. The new sequence data for the rbcL were combined with the extensive batrachospermalean rbcL data available in GenBank. Single gene rbcL results showed the genus Kumanoa to be a well-supported clade, and there was high statistical support for many of the terminal nodes. However, with this gene alone, there was very little support for any of the internal nodes. Analysis of the concatenated data set (rbcL, cox1, and UPA) provided higher statistical support across the tree. The taxa K. vittata and K. amazonensis formed a basal grade, and both were on relatively long branches. Three new species are proposed, K. holtonii, K. gudjewga, and K. novaecaledonensis; K. procarpa var. americana is raised to species level. In addition, the synonymy of K. capensis and K. breviarticulata is proposed, with K. capensis having precedence. Five new combinations are made, bringing the total number of accepted species in Kumanoa to 31. The phylogenetic analyses did not reveal any interpretable biogeographic patterns within the genus (e.g., K. spermatiophora from the tropical oceanic island Maui, Hawaii, was sister to K. faroensis from temperate midcontinental Ohio in North America). Previously hypothesized relationships among groups of species were not substantiated in the phylogenetic analyses, and no intrageneric classification is recommended based on current knowledge.

SYSTEMATICS OF THE BATRACHOSPERMALES (RHODOPHYTA)-A SYNTHESIS(1).

Recent molecular and morphological data necessitate a major taxonomic revision of the Batrachospermales, an order of red algae, distributed in freshwater habitats throughout the world. This article is a synthesis of available information with some targeted additional sequence data, resulting in a few relatively conservative taxonomic changes to begin the process of creating a natural taxonomy for the Batrachospermales. To increase the information content of our taxonomic categories, and in particular to reduce paraphyly, we describe one new genus (Kumanoa) and a new section in Batrachospermum (section Macrospora), and we amend the circumscriptions of the family Batrachospermaceae (to include Lemaneaceae and Psilosiphonaceae), the genus Batrachospermum (to exclude the sections Contorta and Hybrida, raised to genus level as Kumanoa), and the sections Aristata, Helminthoidea, and Batrachospermum of Batrachospermum. We also provide a new name, B. montagnei, for the illegitimate B. guyanense, and recognize an informal paraphyletic grouping of taxa within Batrachospermum, the "Australasica Group." This taxonomic synthesis increases the level of monophyly within the Batrachospermales but minimizes taxonomic change where data are still inadequate.

PHYLOGEOGRAPHY OF BATRACHOSPERMUM MACROSPORUM (BATRACHOSPERMALES, RHODOPHYTA) FROM NORTH AND SOUTH AMERICA(1).

Phylogeographic trends in Batrachospermum macrosporum Mont. were investigated using the mitochondrial intergenic spacer between the cytochrome oxidase subunit 2 and 3 genes (cox2-3). A total of 11 stream segments were sampled with seven in the coastal plain of North America and four in tropical areas of South America. Fifteen thalli were sampled from seven streams, 14 thalli from two streams, and eight thalli from two streams. There were 16 haplotypes detected using 149 individuals. Of the eight haplotypes from locations in North America, all were 334 base pairs (bp) in length, and of those from South America, five were 344 bp, and three were 348 bp. Two individual networks were produced: one for the haplotypes from North America and another for those from South America, and these could not be joined due to the large number of base pair differences. This split between haplotypes from North and South America was confirmed with sequence data of the rbcL gene. There was very little genetic variation among the haplotypes from the North American locations, leading us to hypothesize that these are fairly recent colonization events along the coastal plain. In contrast, there was high variation among haplotypes from South America, and it would appear that the Amazon serves as a center of diversity. We detected considerable variation in haplotypes among streams, but frequently, a single haplotype in an individual stream segment, which is consistent with data from previous studies of other batrachospermalean taxa, may suggest a single colonization event per stream.