Cultivating epizoic diatoms provides insights into the evolution and ecology of both epibionts and hosts.

Our understanding of the importance of microbiomes on large aquatic animals-such as whales, sea turtles and manatees-has advanced considerably in recent years. The latest observations indicate that epibiotic diatom communities constitute diverse, polyphyletic, and compositionally stable assemblages that include both putatively obligate epizoic and generalist species. Here, we outline a successful approach to culture putatively obligate epizoic diatoms without their hosts. That some taxa can be cultured independently from their epizoic habitat raises several questions about the nature of the interaction between these animals and their epibionts. This insight allows us to propose further applications and research avenues in this growing area of study. Analyzing the DNA sequences of these cultured strains, we found that several unique diatom taxa have evolved independently to occupy epibiotic habitats. We created a library of reference sequence data for use in metabarcoding surveys of sea turtle and manatee microbiomes that will further facilitate the use of environmental DNA for studying host specificity in epizoic diatoms and the utility of diatoms as indicators of host ecology and health. We encourage the interdisciplinary community working with marine megafauna to consider including diatom sampling and diatom analysis into their routine practices.

Revision of Ardissoneaceae (Bacillariophyta, Mediophyceae) from Micronesian populations, with descriptions of two new genera, Ardissoneopsis and Grunowago, and new species in Ardissonea, Synedrosphenia and Climacosphenia.

Ardissonea was resurrected from Synedra in 1986 and was included as a genus by Round, Crawford and Mann ("The Diatoms") in its own Family and Order. They commented that there might be several genera involved since the type species of the genus possesses a double-walled structure and other taxa placed in Ardissonea have only a single-walled structure. Two other genera of "big sticks," Toxarium and Climacosphenia, were placed in their own Families and Orders but share many characters with Ardissoneaceae, especially growth from a bifacial annulus. Eighteen taxa (11 new species) from Micronesia were compared with the literature and remnant material from Grunow's Honduras Sargassum sample to address the concepts of Ardissonea and Ardissoneaceae. Phylogenetic and morphological analyses showed three clades within Ardissonea sensu lato: Ardissonea emend. for the double-walled taxa, Synedrosphenia emend. and Ardissoneopsis gen. nov. for single-walled taxa. New species include Ardissoneadensistriata sp. nov.; Synedrospheniabikarensis sp. nov., S.licmophoropsis sp. nov., S.parva sp. nov., and S.recta sp. nov.; Ardissoneopsisfulgicans sp. nov., A.appressata sp. nov., and A.gracilis sp. nov. Transfers include Synedrospheniacrystallina comb. nov. and S.fulgens comb. nov. Synedraundosa, seen for the first time in SEM in Grunow's material, is transferred to Ardissoneopsisundosa comb. nov. Three more genera have similar structure: Toxarium, Climacosphenia and Grunowago gen. nov., erected for Synedrabacillaris and a lanceolate species, G.pacifica sp. nov. Morphological characters of Toxarium in our region support separation of Toxariumhennedyanum and T.undulatum and suggest additional species here and elsewhere. Climacospheniamoniligera was not found but we clarify its characters based on the literature and distinguish C.soulonalis sp. nov. from it. Climacospheniaelongata and a very long, slender C.elegantissima sp. nov., previously identified as C.elongata, were present along with C.scimiter. Morphological and molecular phylogenetics strongly suggested that all these genera belong in one family and we propose to include them in the Ardissoneacae and to reinstate the Order Ardissoneales Round.

Diatom Genus Hyalosira (Rhabdonematales emend.) and Resolution of its Polyphyly in Grammatophoraceae and Rhabdonemataceae with a New Genus, Placosira, and Five New Hyalosira Species.

Hyalosira gene sequences are divided into two clades within different families. We examined authentic material of Hyalosira (isotype material of H. obtusangula, synonymous with H. delicatula) and voucher specimens of published sequences, and pooled our observations of Hyalosira-like taxa from benthic and epizoic habitats in several parts of the globe. The two molecular clades corresponded to two morphological groups, with Hyalosira obtusangula associated with Grammatophoraceae. We emend the description and provide lectotypification for Hyalosira and propose Placosira to encompass the taxa in the other clade, associated with Rhabdonemataceae. We propose that Hyalosira has uniseriate to triseriate striae, sometimes different on valve face and mantle. Copulae in most species had shallow septa, though in one they were moderately deep. All species had girdle bands bearing two rows of areolae separated by a midrib. We name five new species of Hyalosira. Morphologies of taxa in the Placosira clade were superficially similar to Hyalosira but differed in having areolae with ricae, a single row of areolae on the girdle bands, and tubular rimoportulae on the valve-face-mantle junction. Hyalosira hustedtiana Patrick should revert to its original position in Striatella until the appropriate genus can be determined. We emend Rhabdonematales to encompass Rhabdonemataceae, Grammatophoraceae and Tabellariaceae.

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.

Reassessment of the classification of Bryopsidales (Chlorophyta) based on chloroplast phylogenomic analyses.

The Bryopsidales is a morphologically diverse group of mainly marine green macroalgae characterized by a siphonous structure. The order is composed of three suborders - Ostreobineae, Bryopsidineae, and Halimedineae. While previous studies improved the higher-level classification of the order, the taxonomic placement of some genera in Bryopsidineae (Pseudobryopsis and Lambia) as well as the relationships between the families of Halimedineae remains uncertain. In this study, we re-assess the phylogeny of the order with datasets derived from chloroplast genomes, drastically increasing the taxon sampling by sequencing 32 new chloroplast genomes. The phylogenies presented here provided good support for the major lineages (suborders and most families) in Bryopsidales. In Bryopsidineae, Pseudobryopsis hainanensis was inferred as a distinct lineage from the three established families allowing us to establish the family Pseudobryopsidaceae. The Antarctic species Lambia antarctica was shown to be an early-branching lineage in the family Bryopsidaceae. In Halimedineae, we revealed several inconsistent phylogenetic positions of macroscopic taxa, and several entirely new lineages of microscopic species. A new classification scheme is proposed, which includes the merger of the families Pseudocodiaceae, Rhipiliaceae and Udoteaceae into a more broadly circumscribed Halimedaceae, and the establishment of tribes for the different lineages found therein. In addition, the deep-water genus Johnson-sea-linkia, currently placed in Rhipiliopsis, was reinstated based on our phylogeny.

Plastid genome analysis of three Nemaliophycidae red algal species suggests environmental adaptation for iron limited habitats.

The red algal subclass Nemaliophycidae includes both marine and freshwater taxa that contribute to more than half of the freshwater species in Rhodophyta. Given that these taxa inhabit diverse habitats, the Nemaliophycidae is a suitable model for studying environmental adaptation. For this purpose, we characterized plastid genomes of two freshwater species, Kumanoa americana (Batrachospermales) and Thorea hispida (Thoreales), and one marine species Palmaria palmata (Palmariales). Comparative genome analysis identified seven genes (ycf34, ycf35, ycf37, ycf46, ycf91, grx, and pbsA) that were different among marine and freshwater species. Among currently available red algal plastid genomes (127), four genes (pbsA, ycf34, ycf35, ycf37) were retained in most of the marine species. Among these, the pbsA gene, known for encoding heme oxygenase, had two additional copies (HMOX1 and HMOX2) that were newly discovered and were reported from previously red algal nuclear genomes. Each type of heme oxygenase had a different evolutionary history and special modifications (e.g., plastid targeting signal peptide). Based on this observation, we suggest that the plastid-encoded pbsA contributes to the iron controlling system in iron-deprived conditions. Thus, we highlight that this functional requirement may have prevented gene loss during the long evolutionary history of red algal plastid genomes.

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.

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.

Phylogenetic relationships and species circumscription in Trentepohlia and Printzina (Trentepohliales, Chlorophyta).

Subaerial green microalgae represent a polyphyletic complex of organisms, whose genetic diversity is much higher than their simple morphologies suggest. The order Trentepohliales is the only species-rich group of subaerial algae belonging to the class Ulvophyceae and represents an ideal model taxon to investigate evolutionary patterns of these organisms. We studied phylogenetic relationships in two common genera of Trentepohliales (Trentepohlia and Printzina) by separate and combined analyses of the rbcL and 18S rRNA genes. Trentepohlia and Printzina were not resolved as monophyletic groups. Three main clades were recovered in all analyses, but none corresponded to any trentepohlialean genus as defined based on morphological grounds. The rbcL and 18S rRNA datasets provided congruent phylogenetic signals and similar topologies were recovered in single-gene analyses. Analyses performed on the combined 2-gene dataset inferred generally higher nodal support. The results clarified several taxonomic problems and showed that the evolution of these algae has been characterized by considerable morphological convergence. Trentepohlia abietina and T. flava were shown to be separate species from T. aurea; Printzina lagenifera, T. arborum and T. umbrina were resolved as polyphyletic taxa, whose vegetative morphology appears to have evolved independently in separate lineages. Incongruence between phylogenetic relationships and traditional morphological classification was demonstrated, showing that the morphological characters commonly used in the taxonomy of the Trentepohliales are phylogenetically irrelevant.