Diversity of the green macroalgal genus Ulva (Ulvophyceae, Chlorophyta) from the east and gulf coast of the United States based on molecular data.

Ulva (Ulvophyceae, Chlorophyta) is common in intertidal environments and can also be found in freshwater ecosystems. The difficulty to morphologically identify Ulva species due to cryptic diversity and morphological plasticity has caused a taxonomic conundrum. Fortunately, molecular data have begun to unravel a better understanding of its diversity. Here, we present a molecular analysis with 247 samples of Ulva from the Gulf of Mexico and Atlantic USA based on chloroplast (rbcL and tufA) and nuclear (ITS1-5.8S-ITS2) molecular markers. Twenty-four Ulva taxa had previously been reported for this area based on morphology and earlier molecular studies mostly from Northeastern USA and Canada. In this study, sixteen Ulva clades were identified representing 13 named clades and putatively three undescribed species. Only nine of the 24 taxa previously reported for the Western Atlantic were confirmed. Four species were identified for the first time in the U.S. East and Gulf Coast (U. aragoënsis, U. californica, U. meridionalis, and U. tepida). This study provides a foundation for future research on Ulva in this area and reiterates the necessity of using molecular-assisted identifications for this group.

Assessment of littoral algal diversity from the northern Gulf of Mexico using environmental DNA metabarcoding.

Traditional methods for algal biodiversity monitoring are costly and time inefficient because they rely on high-level taxonomic expertise to address species identity problems involving phenotypic plasticity and morphological convergence. These problems are exacerbated in regions such as the Gulf of Mexico, that has a limited history of phycological exploration, but that are economically important or threatened by numerous anthropogenic stressors. Given the high pace of disturbance to natural systems, there is a critical need for expedient and cost-effective tools for the study of benthic algal communities. Here we document the use of environmental DNA metabarcoding, using the partial LSU rDNA and 23S rDNA plastid molecular markers, to elucidate littoral algal diversity in the Northern Gulf of Mexico. We assigned 73.7% of algal OTUs to genus and 59.6% to species ranks. Our current study detected molecular signals for 35 algal/protist species with no previous reports in the Gulf of Mexico, thus providing an important, molecular-validated, baseline of species richness for this region. We also make several bioinformatic recommendations for the efficient use of high-throughput sequence data to assess biological communities.

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.

The chloroplast genomes of Bryopsis plumosa and Tydemania expeditiones (Bryopsidales, Chlorophyta): compact genomes and genes of bacterial origin.

BACKGROUND: Species of Bryopsidales form ecologically important components of seaweed communities worldwide. These siphonous macroalgae are composed of a single giant tubular cell containing millions of nuclei and chloroplasts, and harbor diverse bacterial communities. Little is known about the diversity of chloroplast genomes (cpDNAs) in this group, and about the possible consequences of intracellular bacteria on genome composition of the host. We present the complete cpDNAs of Bryopsis plumosa and Tydemania expeditiones, as well as a re-annotated cpDNA of B. hypnoides, which was shown to contain a higher number of genes than originally published. Chloroplast genomic data were also used to evaluate phylogenetic hypotheses in the Chlorophyta, such as monophyly of the Ulvophyceae (the class in which the order Bryopsidales is currently classified). RESULTS: Both DNAs are circular and lack a large inverted repeat. The cpDNA of B. plumosa is 106,859 bp long and contains 115 unique genes. A 13 kb region was identified with several freestanding open reading frames (ORFs) of putative bacterial origin, including a large ORF (>8 kb) closely related to bacterial rhs-family genes. The cpDNA of T. expeditiones is 105,200 bp long and contains 125 unique genes. As in B. plumosa, several regions were identified with ORFs of possible bacterial origin, including genes involved in mobile functions (transposases, integrases, phage/plasmid DNA primases), and ORFs showing close similarity with bacterial DNA methyltransferases. The cpDNA of B. hypnoides differs from that of B. plumosa mainly in the presence of long intergenic spacers, and a large tRNA region. Chloroplast phylogenomic analyses were largely inconclusive with respect to monophyly of the Ulvophyceae, and the relationship of the Bryopsidales within the Chlorophyta. CONCLUSIONS: The cpDNAs of B. plumosa and T. expeditiones are amongst the smallest and most gene dense chloroplast genomes in the core Chlorophyta. The presence of bacterial genes, including genes typically found in mobile elements, suggest that these have been acquired through horizontal gene transfer, which may have been facilitated by the occurrence of obligate intracellular bacteria in these siphonous algae.

Phylogenetic relationships in Interfilum and Klebsormidium (Klebsormidiophyceae, Streptophyta).

Members of the genus Klebsormidium have cosmopolitan distribution and occur in a very wide range of freshwater and terrestrial habitats. Due to its simple filamentous morphology, this genus represents a taxonomically and systematically complex taxon in which phylogenetic relationships are still poorly understood. The phylogeny of Klebsormidium and closely related taxa was investigated using new ITS rRNA and rbcL sequences generated from 75 strains (isolated from field samples or obtained from culture collections). These sequences were analyzed both as single-marker datasets and in a concatenated dataset. Seven main superclades were observed in the analyses, which included sixteen well-supported clades. Some species of Klebsormidium, including the type species Klebsormidium flaccidum, were polyphyletic. Interfilum was recovered with high statistical support as sister taxon to a clade of Klebsormidium formed mainly by strains identified as K. flaccidum. Whereas some clades could be easily associated with described species, this was not possible for other clades. A new lineage of Klebsormidium, isolated from arid soils in southern Africa and comprising undescribed species, was discovered. Several morphological characters traditionally used for taxonomic purposes were found to have no phylogenetic significance and in some cases showed intra-clade variation. The capacity to form packet-like aggregates (typical of Interfilum), features of the morphology of the chloroplast and the type of habitat were the main phylogenetically relevant characters. Overall, Klebsormidium and Interfilum formed a more diverse algal group than was previously appreciated, with some lineages apparently undergoing active evolutionary radiation; in these lineages the genetic variation observed did not match the morphological and ecological diversity.

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.

The chloroplast genome of the marine green macroalga Ulva fasciata Delile (Ulvophyceae, Chlorophyta).

We present the 96 005 bp circular chloroplast genome (cpDNA) of Ulva fasciata. This cpDNA was ∼4000 bp smaller than the cpDNA of Ulva sp. UNA00071828; however, this cpDNA was AT rich (75.1%) similar to Ulva sp. The U. fasciata cpDNA was also similar in gene content (101 identified genes) compared to Ulva sp., which included 71 protein-coding genes, 3 ribosomal RNAs (rRNAs) and 27 transfer RNAs (tRNAs). Only one tRNA, trnN(AUU), that was present in Ulva sp. was absent in U. fasciata. Five introns were present in the following genes of U. fasciata: petB (2), psbD (1), psaB (1) and rrl (1). Ulva sp. lacked introns in psbD and psaB, and introns present in atpA and psbB in Ulva sp. were absent in the homologous genes of U. fasciata. A gene arrangement comparison of both Ulva species showed that a ∼27 000 bp segment of DNA consisting of genes psbB to trnT(UGU) was inverted. Furthermore, a phylogenetic analysis of 1135 bp of the rbcL gene confirmed that this cpDNA and the previously published mitochondrial genome from this sample were indeed from U. fasciata.

The Plastid Genome in Cladophorales Green Algae Is Encoded by Hairpin Chromosomes.

Virtually all plastid (chloroplast) genomes are circular double-stranded DNA molecules, typically between 100 and 200 kb in size and encoding circa 80-250 genes. Exceptions to this universal plastid genome architecture are very few and include the dinoflagellates, where genes are located on DNA minicircles. Here we report on the highly deviant chloroplast genome of Cladophorales green algae, which is entirely fragmented into hairpin chromosomes. Short- and long-read high-throughput sequencing of DNA and RNA demonstrated that the chloroplast genes of Boodlea composita are encoded on 1- to 7-kb DNA contigs with an exceptionally high GC content, each containing a long inverted repeat with one or two protein-coding genes and conserved non-coding regions putatively involved in replication and/or expression. We propose that these contigs correspond to linear single-stranded DNA molecules that fold onto themselves to form hairpin chromosomes. The Boodlea chloroplast genes are highly divergent from their corresponding orthologs, and display an alternative genetic code. The origin of this highly deviant chloroplast genome most likely occurred before the emergence of the Cladophorales, and coincided with an elevated transfer of chloroplast genes to the nucleus. A chloroplast genome that is composed only of linear DNA molecules is unprecedented among eukaryotes, and highlights unexpected variation in plastid genome architecture.

Chloroplast phylogenomic analyses reveal the deepest-branching lineage of the Chlorophyta, Palmophyllophyceae class. nov.

The green plants (Viridiplantae) are an ancient group of eukaryotes comprising two main clades: the Chlorophyta, which includes a wide diversity of green algae, and the Streptophyta, which consists of freshwater green algae and the land plants. The early-diverging lineages of the Viridiplantae comprise unicellular algae, and multicellularity has evolved independently in the two clades. Recent molecular data have revealed an unrecognized early-diverging lineage of green plants, the Palmophyllales, with a unique form of multicellularity, and typically found in deep water. The phylogenetic position of this enigmatic group, however, remained uncertain. Here we elucidate the evolutionary affinity of the Palmophyllales using chloroplast genomic, and nuclear rDNA data. Phylogenetic analyses firmly place the palmophyllalean Verdigellas peltata along with species of Prasinococcales (prasinophyte clade VI) in the deepest-branching clade of the Chlorophyta. The small, compact and intronless chloroplast genome (cpDNA) of V. peltata shows striking similarities in gene content and organization with the cpDNAs of Prasinococcales and the streptophyte Mesostigma viride, indicating that cpDNA architecture has been extremely well conserved in these deep-branching lineages of green plants. The phylogenetic distinctness of the Palmophyllales-Prasinococcales clade, characterized by unique ultrastructural features, warrants recognition of a new class of green plants, Palmophyllophyceae class. nov.

The complete chloroplast and mitochondrial genomes of the green macroalga Ulva sp. UNA00071828 (Ulvophyceae, Chlorophyta).

Sequencing mitochondrial and chloroplast genomes has become an integral part in understanding the genomic machinery and the phylogenetic histories of green algae. Previously, only three chloroplast genomes (Oltmannsiellopsis viridis, Pseudendoclonium akinetum, and Bryopsis hypnoides) and two mitochondrial genomes (O. viridis and P. akinetum) from the class Ulvophyceae have been published. Here, we present the first chloroplast and mitochondrial genomes from the ecologically and economically important marine, green algal genus Ulva. The chloroplast genome of Ulva sp. was 99,983 bp in a circular-mapping molecule that lacked inverted repeats, and thus far, was the smallest ulvophycean plastid genome. This cpDNA was a highly compact, AT-rich genome that contained a total of 102 identified genes (71 protein-coding genes, 28 tRNA genes, and three ribosomal RNA genes). Additionally, five introns were annotated in four genes: atpA (1), petB (1), psbB (2), and rrl (1). The circular-mapping mitochondrial genome of Ulva sp. was 73,493 bp and follows the expanded pattern also seen in other ulvophyceans and trebouxiophyceans. The Ulva sp. mtDNA contained 29 protein-coding genes, 25 tRNA genes, and two rRNA genes for a total of 56 identifiable genes. Ten introns were annotated in this mtDNA: cox1 (4), atp1 (1), nad3 (1), nad5 (1), and rrs (3). Double-cut-and-join (DCJ) values showed that organellar genomes across Chlorophyta are highly rearranged, in contrast to the highly conserved organellar genomes of the red algae (Rhodophyta). A phylogenomic investigation of 51 plastid protein-coding genes showed that Ulvophyceae is not monophyletic, and also placed Oltmannsiellopsis (Oltmannsiellopsidales) and Tetraselmis (Chlorodendrophyceae) closely to Ulva (Ulvales) and Pseudendoclonium (Ulothrichales).

The mitochondrial genome of Grateloupia taiwanensis (Halymeniaceae, Rhodophyta) and comparative mitochondrial genomics of red algae.

Although red algae are economically highly valuable for their gelatinous cell wall compounds as well as being integral parts of marine benthic habitats, very little genome data are currently available. We present mitochondrial genome sequence data from the red alga Grateloupia taiwanensis S.-M. Lin & H.-Y. Liang. Comprising 28,906 nucleotide positions, the mitochondrial genome contig contains 25 protein-coding genes and 24 transfer RNA genes. It is highly similar to other red algal genomes in gene content as well as overall structure. An intron in the cox1 gene was found to be shared by G. taiwanensis and Grateloupia angusta (Okamura) S. Kawaguchi & H. W. Wang. We also used whole-genome alignments to compare G. taiwanensis to different groups of red algae, and these results are consistent with the currently accepted phylogeny of Rhodophyta.

The plastid genome of the red macroalga Grateloupia taiwanensis (Halymeniaceae).

The complete plastid genome sequence of the red macroalga Grateloupia taiwanensis S.-M.Lin & H.-Y.Liang (Halymeniaceae, Rhodophyta) is presented here. Comprising 191,270 bp, the circular DNA contains 233 protein-coding genes and 29 tRNA sequences. In addition, several genes previously unknown to red algal plastids are present in the genome of G. taiwanensis. The plastid genomes from G. taiwanensis and another florideophyte, Gracilaria tenuistipitata var. liui, are very similar in sequence and share significant synteny. In contrast, less synteny is shared between G. taiwanensis and the plastid genome representatives of Bangiophyceae and Cyanidiophyceae. Nevertheless, the gene content of all six red algal plastid genomes here studied is highly conserved, and a large core repertoire of plastid genes can be discerned in Rhodophyta.

DISTRIBUTION, MORPHOLOGY, AND PHYLOGENY OF KLEBSORMIDIUM (KLEBSORMIDIALES, CHAROPHYCEAE) IN URBAN ENVIRONMENTS IN EUROPE(1).

Klebsormidium is a cosmopolitan genus of green algae, widespread in terrestrial and freshwater habitats. The classification of Klebsormidium is entirely based on morphological characters, and very little is understood about its phylogeny at the species level. We investigated the diversity and phylogenetic relationships of Klebsormidium in urban habitats in Europe by a combination of approaches including examination of field-collected material, culture experiments conducted in many different combinations of factors, and phylogenetic analyses of the rbcL gene. Klebsormidium in European cities mainly occurs at the base of old walls, where it may produce green belts up to several meters in extent. Specimens from different cities showed a great morphological uniformity, consisting of long filaments 6-9 µm in width, with thin-walled cylindrical cells and smooth wall, devoid of false branches, H-shaped pieces, and biseriate parts. Conversely, the rbcL phylogeny showed a higher genetic diversity than expected from morphology. The strains were separated in four different clades supported by high bootstrap values and posterior probabilities. In culture, these clades differed in several characters, such as production of a superficial hydro-repellent layer, tendency to break into short fragments, and inducibility of zoosporulation. On the basis of the taxonomic information available in the literature, most strains could not be identified unambiguously at the species level. The rbcL phylogeny showed no correspondence with classification based on morphology and suggested that the identity of many species, in particular the type species K. flaccidum (kütz.) P.C. Silva, Mattox et W. H. Blackw., needs critical reassessment.

Molecular systematics of the subaerial green algal order Trentepohliales: an assessment based on morphological and molecular data.

The order Trentepohliales is a widespread group of terrestrial green algae. As currently circumscribed, it includes six genera (Cephaleuros, Phycopeltis, Physolinum, Printzina, Stomatochroon and Trentepohlia), the phylogenetic affinities of which are poorly understood. Sequences for the small subunit rRNA gene (18S rDNA) were obtained for several representatives of the order and phylogenetic analyses based on these sequences were compared with the traditional system of classification based on morphological characters. The results indicate that the morphological criteria used for the circumscription of genera and species of the Trentepohliales do not match phylogenetic patterns. Whereas isolates of Cephaleuros form a well-supported monophyletic clade, Trentepohlia is polyphyletic and strains of Trentepohlia, Phycopeltis, Printzina and Physolinum are mixed together in several different lineages. A reassessment of the generic boundaries and a narrower circumscription of some genera (in particular Trentepohlia) will be necessary. Subcuticular habit, heteromorphic life history and occurrence of zoosporangia in clusters are shown to be phylogenetically significant features, whereas other characters commonly used for separation of species and genera (shape and size of the cells, prostrate/erect habit, substratum colonized) have no phylogenetic relevance.

Morphology and phylogenetic position of Spongiochrysis hawaiiensis gen. et sp. nov., the first known terrestrial member of the order Cladophorales (Ulvophyceae, Chlorophyta).

Spongiochrysis hawaiiensis gen. et sp. nov. is described from material collected at two sites on O'ahu, Hawaiian Islands. This alga produced golden-yellow crusts on the bark of Casuarina trees and consisted of globular cells with an axial stellate chloroplast. The only form of reproduction was a specialized type of autosporulation in which a budding-like division of the mother cell produced daughter cells of different sizes. Phylogenetic analyses of the 18S rRNA gene showed that Spongiochrysis hawaiiensis is a member of the freshwater clade of the Cladophorales/Siphonocladales lineage in the green algal class Ulvophyceae. On the basis of its unicellular habit and terrestrial habitat, this species is well differentiated from all other members of the same lineage and can be considered as the first known successful step of a member of the order Cladophorales into terrestrial habitats. The implications of the description of this species with regard to both green algal evolution and conservation of little-known tropical habitats are discussed. The holotype specimen of Spongiochrysis hawaiiensis is GALW015489 and isotype specimens have also been deposited in the BISH and BM herbaria.

Phylogenetic affinities of the Trentepohliales inferred from small-subunit rDNA.

Phylogenetic analyses of the nuclear-encoded small-subunit rDNA sequences from taxa representing all of the major lineages of green algae, including new sequences for the Trentepohliales, consistently indicated that the subaerial Trentepohliales are closely related to ulvophycean marine green algae, particularly to the siphonous and hemisiphonous orders. The presence of phragmoplast-type cytokinesis in the order Trentepohliales remains enigmatic, and it is interesting that this type of cell division is associated with terrestrial (subaerial) habits.

Phragmoplastin, green algae and the evolution of cytokinesis.

Phragmoplast-mediated cell division characterizes the land plants in the streptophyte lineage and some species of the green algal orders Coleochaetales, Charales and Zygnematales that are basal to that lineage. This type of cell division is generally not found in the other green plant lineage, the chlorophyte algae. A well-developed phragmoplast-type cell division has been documented, however, in two subaerial green algae (Cephaleuros parasiticus and Trentepohlia odorata) belonging to the order Trentepohliales--an order that molecular sequence data place unequivocally within the chlorophytes rather than streptophytes. Is the phragmoplast-mediated cell division of the Trentepohliales a case of homology or non-homology? To gain more insight into this question, we are exploring the potential phylogenetic information inferred from gene sequences of phragmoplastin, a dynamin-like protein that has been associated with cell-plate formation during phragmoplast-mediated cytokinesis in land plants. Primers for green algae were designed based on an available phragmoplastin sequence from soybean and yielded PCR amplifications from the trentepohlialean green algae Trentepohlia and Cephaleuros and the leafy liverwort Bazzania: These are the first published data for phragmoplastins in algae and liverworts. Analysis of phragmoplastin gene sequences in chlorophyte and streptophyte green algae may help to chart the evolution of the development of the phragmoplast.