Contrasting patterns of genetic structure and phylogeography in the marine agarophytes Gelidiophycus divaricatus and G. freshwateri (Gelidiales, Rhodophyta) from East Asia.

The evolutionary and population demographic history of marine red algae in East Asia is poorly understood. Here, we reconstructed the phylogeographies of two upper intertidal species endemic to East Asia, Gelidiophycus divaricatus and G. freshwateri. Phylogenetic and phylogeographic inferences of 393 mitochondrial cox1, 128 plastid rbcL, and 342 nuclear ITS2 sequences were complemented with ecological niche models. Gelidiophycus divaricatus, a southern species adapted to warm water, is characterized by a high genetic diversity and a strong geographical population structure, characteristic of stable population sizes and sudden reduction to recent expansion. In contrast, G. freshwateri, a northern species adapted to cold temperate conditions, is genetically relatively homogeneous with a shallow population structure resulting from steady population growth and recent equilibrium. The overlap zone of the two species roughly matches summer and winter isotherms, indicating that surface seawater temperature is a key feature influencing species range. Unidirectional genetic introgression was detected at two sites on Jeju Island where G. divaricatus was rare while G. freshwateri was common, suggesting the occurrence of asymmetric natural hybrids, a rarely reported event for rhodophytes. Our results illustrate that Quaternary climate oscillations have left strong imprints on the current day genetic structure and highlight the importance of seawater temperature and sea level change in driving speciation in upper intertidal seaweed species.

Analysis of the Draft Genome of the Red Seaweed Gracilariopsis chorda Provides Insights into Genome Size Evolution in Rhodophyta.

Red algae (Rhodophyta) underwent two phases of large-scale genome reduction during their early evolution. The red seaweeds did not attain genome sizes or gene inventories typical of other multicellular eukaryotes. We generated a high-quality 92.1 Mb draft genome assembly from the red seaweed Gracilariopsis chorda, including methylation and small (s)RNA data. We analyzed these and other Archaeplastida genomes to address three questions: 1) What is the role of repeats and transposable elements (TEs) in explaining Rhodophyta genome size variation, 2) what is the history of genome duplication and gene family expansion/reduction in these taxa, and 3) is there evidence for TE suppression in red algae? We find that the number of predicted genes in red algae is relatively small (4,803-13,125 genes), particularly when compared with land plants, with no evidence of polyploidization. Genome size variation is primarily explained by TE expansion with the red seaweeds having the largest genomes. Long terminal repeat elements and DNA repeats are the major contributors to genome size growth. About 8.3% of the G. chorda genome undergoes cytosine methylation among gene bodies, promoters, and TEs, and 71.5% of TEs contain methylated-DNA with 57% of these regions associated with sRNAs. These latter results suggest a role for TE-associated sRNAs in RNA-dependent DNA methylation to facilitate silencing. We postulate that the evolution of genome size in red algae is the result of the combined action of TE spread and the concomitant emergence of its epigenetic suppression, together with other important factors such as changes in population size.

Phylogenetic relationships and biogeography of Ptilophora (Gelidiales, Rhodophyta) with descriptions of P. aureolusa, P. malagasya, and P. spongiophila from Madagascar.

The genus Ptilophora currently includes 16 species occurring mostly in subtidal habitats of the Indo-Pacific Ocean, but its global diversity and biogeography are poorly understood. We analyzed mitochondrial cox1, plastid rbcL and plastid psbA sequences from specimens collected in southern Madagascar during the 2010 Atimo Vatae expedition and studied their morphologies. Both morphological and molecular data sets demonstrated the presence of five species in southern Madagascar: Ptilophora hildebrandtii, P. pterocladioides, and three new species described here, P. aureolusa, P. malagasya, and P. spongiophila. Ptilophora aureolusa is distinguished by its compound pinnae with uniformly spaced pinnules. Ptilophora malagasya has an indistinct midrib and irregularly spaced pinnules. Ptilophora spongiophila, heavily coated with sponges, has cylindrical to flattened main axes, lateral and surface proliferations, and spatulate tetrasporangial sori. The species of Ptilophora found in Madagascar are endemic, except P. hildebrandtii, which also occurs in eastern Africa. Ptilophora comprises four phylogenetic groups that map to eastern Australia, Japan, western Australia/Southeast Asia/Madagascar/eastern Africa, and Madagascar/eastern Africa/Aegean Sea. Biogeographical analysis revealed that the ancestor of Ptilophora originated in Australia, but most of the species radiated from Madagascar.

The complete mitochondrial genome of the horned lizard Phrynosoma blainvillii (Squamata: Phrynosomatidae) from California, USA.

Analysis of Phrynosoma blainvillii Gray from Marina, Monterey County, California, using 150 bp paired-end Illumina sequences (Illumina, San Diego, CA) resulted in the assembly of its complete mitogenome. The mitogenome is 16,946 bp in length and contains a putative origin of light strand replication (OL), control region, 22 tRNA, 2 rRNA, and 13 protein-coding genes. Its content and organization are similar to other Squamata. Phylogenetic analysis of P. blainvillii resolves it in a clade with P. sherbrookei Nieto-Montes de Oca, Arenas-Moreno, Beltrán-Sánchez & Leaché, sister in position to Uma notata Baird. Mitochondrial marker analysis of P. blainvillii from Marina shows that it belongs to a coastal Santa Lucia Mountain Range haplogroup that is distinct from other populations of P. blainvillii in California.

Increased potential for wound activated production of Prostaglandin E2 and related toxic compounds in non-native populations of Gracilaria vermiculophylla.

The capacity of the East Asian seaweed Gracilaria vermiculophylla ("Ogonori") for production of prostaglandin E2 from arachidonic acid occasionally causes food poisoning after ingestion. During the last two decades the alga has been introduced to Europe and North America. Non-native populations have been shown to be generally less palatable to marine herbivores than native populations. We hypothesized that the difference in palatability among populations could be due to differences in the algal content of prostaglandins. We therefore compared the capacity for wound-activated production of prostaglandins and other eicosatetraenoid oxylipins among five native populations in East Asia and seven non-native populations in Europe and NW Mexico, using a targeted metabolomics approach. In two independent experiments non-native populations exhibited a significant tendency to produce more eicosatetraenoids than native populations after acclimation to identical conditions and subsequent artificial wounding. Fourteen out of 15 eicosatetraenoids that were detected in experiment I and all 19 eicosatetraenoids that were detected in experiment II reached higher mean concentrations in non-native than in native specimens. Wounding of non-native specimens resulted on average in 390% more 15-keto-PGE2, in 90% more PGE2, in 37% more PGA2 and in 96% more 7,8-di-hydroxy-eicosatetraenoic acid than wounding of native specimens. Not only PGE2, but also PGA2 and dihydroxylated eicosatetraenoic acid are known to deter various biological enemies of G. vermiculophylla that cause tissue or cell wounding, and in the present study the latter two compounds also repelled the mesograzer Littorina brevicula. Non-native populations of G. vermiculophylla are thus more defended against herbivory than native populations. This increased capacity for activated chemical defense may have contributed to their invasion success and at the same time it poses an elevated risk for human food safety.

Mitogenomes from type specimens, a genotyping tool for morphologically simple species: ten genomes of agar-producing red algae.

DNA sequences from type specimens provide independent, objective characters that enhance the value of type specimens and permit the correct application of species names to phylogenetic clades and specimens. We provide mitochondrial genomes (mitogenomes) from archival type specimens of ten species in agar-producing red algal genera Gelidium and Pterocladiella. The genomes contain 43-44 genes, ranging in size from 24,910 to 24,970 bp with highly conserved gene synteny. Low Ka/Ks ratios of apocytochrome b and cytochrome oxidase genes support their utility as markers. Phylogenies of mitogenomes and cox1+rbcL sequences clarified classification at the genus and species levels. Three species formerly in Gelidium and Pterocladia are transferred to Pterocladiella: P. media comb. nov., P. musciformis comb. nov., and P. luxurians comb. and stat. nov. Gelidium sinicola is merged with G. coulteri because they share identical cox1 and rbcL sequences. We describe a new species, Gelidium millariana sp. nov., previously identified as G. isabelae from Australia. We demonstrate that mitogenomes from type specimens provide a new tool for typifying species in the Gelidiales and that there is an urgent need for analyzing mitogenomes from type specimens of red algae and other morphologically simple organisms for insight into their nomenclature, taxonomy and evolution.

A novel phylogeny of the Gelidiales (Rhodophyta) based on five genes including the nuclear CesA, with descriptions of Orthogonacladia gen. nov. and Orthogonacladiaceae fam. nov.

Although the Gelidiales are economically important marine red algae producing agar and agarose, the phylogeny of this order remains poorly resolved. The present study provides a molecular phylogeny based on a novel marker, nuclear-encoded CesA, plus plastid-encoded psaA, psbA, rbcL, and mitochondria-encoded cox1 from subsets of 107 species from all ten genera within the Gelidiales. Analyses of individual and combined datasets support the monophyly of three currently recognized families, and reveal a new clade. On the basis of these results, the new family Orthogonacladiaceae is described to accommodate Aphanta and a new genus Orthogonacladia that includes species previously classified as Gelidium madagascariense and Pterocladia rectangularis. Acanthopeltis is merged with Gelidium, which has nomenclatural priority. Nuclear-encoded CesA was found to be useful for improving the resolution of phylogenetic relationships within the Gelidiales and is likely to be valuable for the inference of phylogenetic relationship among other red algal taxa.

Reconstructing the complex evolutionary history of mobile plasmids in red algal genomes.

The integration of foreign DNA into algal and plant plastid genomes is a rare event, with only a few known examples of horizontal gene transfer (HGT). Plasmids, which are well-studied drivers of HGT in prokaryotes, have been reported previously in red algae (Rhodophyta). However, the distribution of these mobile DNA elements and their sites of integration into the plastid (ptDNA), mitochondrial (mtDNA), and nuclear genomes of Rhodophyta remain unknown. Here we reconstructed the complex evolutionary history of plasmid-derived DNAs in red algae. Comparative analysis of 21 rhodophyte ptDNAs, including new genome data for 5 species, turned up 22 plasmid-derived open reading frames (ORFs) that showed syntenic and copy number variation among species, but were conserved within different individuals in three lineages. Several plasmid-derived homologs were found not only in ptDNA but also in mtDNA and in the nuclear genome of green plants, stramenopiles, and rhizarians. Phylogenetic and plasmid-derived ORF analyses showed that the majority of plasmid DNAs originated within red algae, whereas others were derived from cyanobacteria, other bacteria, and viruses. Our results elucidate the evolution of plasmid DNAs in red algae and suggest that they spread as parasitic genetic elements. This hypothesis is consistent with their sporadic distribution within Rhodophyta.

Divergence time estimates and the evolution of major lineages in the florideophyte red algae.

The Florideophyceae is the most abundant and taxonomically diverse class of red algae (Rhodophyta). However, many aspects of the systematics and divergence times of the group remain unresolved. Using a seven-gene concatenated dataset (nuclear EF2, LSU and SSU rRNAs, mitochondrial cox1, and plastid rbcL, psaA and psbA genes), we generated a robust phylogeny of red algae to provide an evolutionary timeline for florideophyte diversification. Our relaxed molecular clock analysis suggests that the Florideophyceae diverged approximately 943 (817-1,049) million years ago (Ma). The major divergences in this class involved the emergence of Hildenbrandiophycidae [ca. 781 (681-879) Ma], Nemaliophycidae [ca. 661 (597-736) Ma], Corallinophycidae [ca. 579 (543-617) Ma], and the split of Ahnfeltiophycidae and Rhodymeniophycidae [ca. 508 (442-580) Ma]. Within these clades, extant diversity reflects largely Phanerozoic diversification. Divergences within Florideophyceae were accompanied by evolutionary changes in the carposporophyte stage, leading to a successful strategy for maximizing spore production from each fertilization event. Our research provides robust estimates for the divergence times of major lineages within the Florideophyceae. This timeline was used to interpret the emergence of key morphological innovations that characterize these multicellular red algae.

Mitogenome of Mytilus trossulus (Mytilidae, Bivalvia) isolated from a 1920 herbarium specimen.

DNA was extracted from a red algal herbarium specimen collected in 1920 and subjected to next generation sequencing. Here we report the assembly of the mitogenome of a marine mussel, Mytilus trossulus, deciphered from this plant museum specimen. The mitogenome is 16,744 bp in length, contains 38 genes, and is more similar to other M. trossulus reported from the Baltic Sea. The data show that in addition to plant DNA, herbarium specimens also contain genetic information from invertebrates that may be valuable for genomic, population and phylogenetic studies of animals.

Highly Conserved Mitochondrial Genomes among Multicellular Red Algae of the Florideophyceae.

Two red algal classes, the Florideophyceae (approximately 7,100 spp.) and Bangiophyceae (approximately 193 spp.), comprise 98% of red algal diversity in marine and freshwater habitats. These two classes form well-supported monophyletic groups in most phylogenetic analyses. Nonetheless, the interordinal relationships remain largely unresolved, in particular in the largest subclass Rhodymeniophycidae that includes 70% of all species. To elucidate red algal phylogenetic relationships and study organelle evolution, we determined the sequence of 11 mitochondrial genomes (mtDNA) from 5 florideophycean subclasses. These mtDNAs were combined with existing data, resulting in a database of 25 florideophytes and 12 bangiophytes (including cyanidiophycean species). A concatenated alignment of mt proteins was used to resolve ordinal relationships in the Rhodymeniophycidae. Red algal mtDNA genome comparisons showed 47 instances of gene rearrangement including 12 that distinguish Bangiophyceae from Hildenbrandiophycidae, and 5 that distinguish Hildenbrandiophycidae from Nemaliophycidae. These organelle data support a rapid radiation and surprisingly high conservation of mtDNA gene syntheny among the morphologically divergent multicellular lineages of Rhodymeniophycidae. In contrast, we find extensive mitochondrial gene rearrangements when comparing Bangiophyceae and Florideophyceae and multiple examples of gene loss among the different red algal lineages.

Complete mitochondrial genome of the agarophyte red alga Gelidium vagum (Gelidiales).

We describe the first complete mitochondrial genome of Gelidium vagum (Gelidiales) (24,901 bp, 30.4% GC content), an agar-producing red alga. The circular mitochondrial genome contains 43 genes, including 23 protein-coding, 18 tRNA and 2 rRNA genes. All the protein-coding genes have a typical ATG start codon. No introns were found. Two genes, secY and rps12, were overlapped by 41 bp.

Complete mitochondrial genome of the marine red alga Grateloupia angusta (Halymeniales).

The first complete mitochondrial genome (27,943 bp) of the halymenialean red alga, Grateloupia angusta, was sequenced. The circular mitogenome contains 47 genes, including 26 protein-coding, 19 tRNA and 2 rRNA genes. The rps3 and rpl16 genes are overlapped by 7 bp. The majority of protein-coding genes (17 of 26 genes) use typical ATG start codon, however, 9 genes use alternative start codons such as ATT, AAT, TTA and TTG. The G. angusta mitogenome shows a significant difference to that of Chondrus crispus (Gigartinales) in gene synteny and tRNA compositions.

Genetic diversity and haplotype distribution of Pachymeniopsis gargiuli sp. nov. and P. lanceolata (Halymeniales, Rhodophyta) in Korea, with notes on their non-native distributions.

The red alga Pachymeniopsis lanceolata, formerly known as Grateloupia lanceolata, is a component of the native algal flora of northeast Asia and has been introduced to European and North American waters. It has been confused with a cryptic species collected from Korea and Italy. Our analyses of rbcL, cox3 and ITS from P. lanceolata and this cryptic species has revealed two distinct entities, forming a clade, which were clearly separated from its congeners and positioned with other Asian species. Here, we describe the cryptic species as P. gargiuli sp. nov., a species that differs from others by molecular sequence and subtle anatomical characters. We hypothesize that P. gargiuli may have been recently dispersed by anthropogenic vectors, possibly at or near the same time as was P. lanceolata. Our cox3 data set revealed that one haplotype of P. gargiuli, shared between Korea and Italy, and two haplotypes of P. lanceolata, commonly occurring in Korea and USA, are invasive haplotypes. This is the first report of the utility of the mitochondrial coding cox3 sequences in red algae.

Supermatrix data highlight the phylogenetic relationships of photosynthetic stramenopiles.

Molecular data had consistently recovered monophyletic classes for the heterokont algae, however, the relationships among the classes had remained only partially resolved. Furthermore, earlier studies did not include representatives from all taxonomic classes. We used a five-gene (nuclear encoded SSU rRNA; plastid encoded rbcL, psaA, psbA, psbC) analysis with a subset of 89 taxa representing all 16 heterokont classes to infer a phylogenetic tree. There were three major clades. The Aurearenophyceae, Chrysomerophyceae, Phaeophyceae, Phaeothamniophyceae, Raphidophyceae, Schizocladiophyceae and Xanthophyceae formed the SI clade. The Chrysophyceae, Eustigmatophyceae, Pinguiophyceae, Synchromophyceae and Synurophyceae formed the SII clade. The Bacillariophyceae, Bolidophyceae, Dictyochophyceae and Pelagophyceae formed the SIII clade. These three clades were also found in a ten-gene analysis. The approximately unbiased test rejected alternative hypotheses that forced each class into either of the other two clades. Morphological and biochemical data were not available for all 89 taxa, however, existing data were consistent with the molecular phylogenetic tree, especially for the SIII clade.

A NEW AGAROPHYTE SPECIES, GELIDIUM EUCORNEUM SP. NOV. (GELIDIALES, RHODOPHYTA), BASED ON MOLECULAR AND MORPHOLOGICAL DATA(1).

Gelidium is an economically and ecologically important agar-producing genus. Although the taxonomy of Gelidium has been the focus of many published studies, there is still a need to reevaluate species-level diversity. Herein, we describe Gelidium eucorneum sp. nov. based on specimens collected off Geojedo on the southern coast of Korea. G. eucorneum is distinguished by cartilaginous thalli with brush-like haptera, rhizoidal filaments concentrated in the medulla, and globose cytocarps that are horned with multiple determinate branchlets. The species occurs in wave-exposed intertidal sites, sometimes in association with other mat-forming algae. Phylogenetic analyses (rbcL, psaA, and cox1) reveal that G. eucorneum is unique and clearly distinct from other species of the genus. The clade containing Gelidium vagum and Acanthopeltis longiramulosa was resolved as a sister group to G. eucorneum. We suggest that the diverse morphologies of G. eucorneum, G. vagum, and Acanthopeltis developed from a common ancestor in East Asian waters. This study shows that even in well-studied areas, more agarophyte species are to be added to the world inventory of red algae.

STUDIES ON ULTRASTRUCTURE AND THREE-GENE PHYLOGENY OF THE GENUS MALLOMONAS (SYNUROPHYCEAE)(1).

The genus Mallomonas, a common and often abundant member of the planktic community in many freshwater habitats worldwide, consists of 180 species divided into 19 sections and 23 series. Classification of species is based largely on ultrastructural characteristics of the siliceous scales and bristles that collectively form a highly organized covering over the cell. However, the relative importance of the different siliceous features of the scales, such as the dome, V rib, and secondary structures, as well as the different types of scales, in understanding the evolution and phylogeny of the genus is little known. In this study, we investigated the scale and bristle ultrastructure, along with sequences of three genes, for 19 isolates (18 species) of Mallomonas (18 isolates were from Korean habitats). The isolates represented nine of the 19 sections. Sequences for both the nuclear SSU and LSU rDNA and plastid LSU of RUBISCO (rbcL) genes for each of the 19 Mallomonas isolates and four outgroups were determined. Bayesian and maximum-likelihood (ML) analyses of the data revealed that Mallomonas consists of two strongly supported clades. Mallomonas bangladeshica (E. Takah. et T. Hayak.) Siver et A. P. Wolfe was at the base of the first clade that included taxa from the sections Planae and Heterospinae, both of which lack a V rib on the shield of the scales. Our results indicated that the sections Planae and Heterospinae should be combined. The second clade, with Mallomonas insignis Penard and Mallomonas punctifera Korshikov at the base, contained taxa from the sections Mallomonas, Striatae, Akrokomae, Annulatae, Torquatae, Punctiferae, and Insignes, all of which have V ribs or well-developed marginal ribs on the scales. Sister relationships between Mallomonas and Striatae were strongly supported, but interrelations among the remaining sections were not resolved, probably due to inclusion of too few species. Our results suggest that the current classification of the genus Mallomonas at the section level will require some revision. Additional species will need to be added in future analyses.

Annual variation of Microcystis genotypes and their potential toxicity in water and sediment from a eutrophic reservoir.

The relative genetic diversity of microcystin-producing Microcystis in the water and sediment of the Daechung Reservoir, Korea, was investigated over an entire year, including the cyanobacterial bloom season. The cells of potentially toxic Microcystis strains containing mcyJ genotypes and cells containing the genus-specific cpcBA gene were quantified by a real-time PCR. The ratio of cells with mcyJ genotypes to the total Microcystis population in the water body was the highest (68.3%) in August when the cyanobacterial bloom reached its peak and the microcystin concentration in the water began to increase. A denaturing gradient gel electrophoresis profile analysis of the mcyJ genotypes performed to monitor any changes in the toxic Microcystis population showed the appearance of new genotypes and the disappearance of existing genotypes in the reservoir water collected during the summer months, when compared with the profile for the samples collected in spring and autumn. However, very little change was observed over the course of the year as regards the population diversity of the sediment samples.

Differential gene retention in plastids of common recent origin.

The cyanobacterium-derived plastids of algae and plants have supported the diversification of much of extant eukaryotic life. Inferences about early events in plastid evolution must rely on reconstructing events that occurred over a billion years ago. In contrast, the photosynthetic amoeba Paulinella chromatophora provides an exceptional model to study organelle evolution in a prokaryote-eukaryote (primary) endosymbiosis that occurred approximately 60 mya. Here we sequenced the plastid genome (0.977 Mb) from the recently described Paulinella FK01 and compared the sequence with the existing data from the sister taxon Paulinella M0880/a. Alignment of the two plastid genomes shows significant conservation of gene order and only a handful of minor gene rearrangements. Analysis of gene content reveals 66 differential gene losses that appear to be outright gene deletions rather than endosymbiotic gene transfers to the host nuclear genome. Phylogenomic analysis validates the plastid ancestor as a member of the Synechococcus-Prochlorococcus group, and the cyanobacterial provenance of all plastid genes suggests that these organelles were not targets of interphylum gene transfers after endosymbiosis. Inspection of 681 DNA alignments of protein-encoding genes shows that the vast majority have dN/dS ratios <<1, providing evidence for purifying selection. Our study demonstrates that plastid genomes in sister taxa are strongly constrained by selection but follow distinct trajectories during the earlier phases of organelle evolution.

Complex phylogeographic patterns in the freshwater alga Synura provide new insights into ubiquity vs. endemism in microbial eukaryotes.

The global distribution, abundance, and diversity of microscopic freshwater algae demonstrate an ability to overcome significant barriers such as dry land and oceans by exploiting a range of biotic and abiotic colonization vectors. If these vectors are considered unlimited and colonization occurs in proportion to population size, then globally ubiquitous distributions are predicted to arise. This model contrasts with observations that many freshwater microalgal taxa possess true biogeographies. Here, using a concatenated multigene data set, we study the phylogeography of the freshwater heterokont alga Synura petersenii sensu lato. Our results suggest that this Synura morphotaxon contains both cosmopolitan and regionally endemic cryptic species, co-occurring in some cases, and masked by a common ultrastructural morphology. Phylogenies based on both proteins (seven protein-coding plastid and mitochondrial genes) and DNA (nine genes including ITS and 18S rDNA) reveal pronounced biogeographic delineations within phylotypes of this cryptic species complex while retaining one clade that is globally distributed. Relaxed molecular clock calculations, constrained by fossil records, suggest that the genus Synura is considerably older than currently proposed. The availability of tectonically relevant geological time (107-108 years) has enabled the development of the observed, complex biogeographic patterns. Our comprehensive analysis of freshwater algal biogeography suggests that neither ubiquity nor endemism wholly explains global patterns of microbial eukaryote distribution and that processes of dispersal remain poorly understood.