Diurnal Rhythms in the Red Seaweed Gracilariopsis chorda are Characterized by Unique Regulatory Networks of Carbon Metabolism.

Cellular and physiological cycles are driven by endogenous pacemakers, the diurnal and circadian rhythms. Key functions such as cell cycle progression and cellular metabolism are under rhythmic regulation, thereby maintaining physiological homeostasis. The photoreceptors phytochrome and cryptochrome, in response to light cues, are central input pathways for physiological cycles in most photosynthetic organisms. However, among Archaeplastida, red algae are the only taxa that lack phytochromes. Current knowledge about oscillatory rhythms is primarily derived from model species such as Arabidopsis thaliana and Chlamydomonas reinhardtii in the Viridiplantae, whereas little is known about these processes in other clades of the Archaeplastida, such as the red algae (Rhodophyta). We used genome-wide expression profiling of the red seaweed Gracilariopsis chorda and identified 3,098 rhythmic genes. Here, we characterized possible cryptochrome-based regulation and photosynthetic/cytosolic carbon metabolism in this species. We found a large family of cryptochrome genes in G. chorda that display rhythmic expression over the diurnal cycle and may compensate for the lack of phytochromes in this species. The input pathway gates regulatory networks of carbon metabolism which results in a compact and efficient energy metabolism during daylight hours. The system in G. chorda is distinct from energy metabolism in most plants, which activates in the dark. The green lineage, in particular, land plants, balance water loss and CO2 capture in terrestrial environments. In contrast, red seaweeds maintain a reduced set of photoreceptors and a compact cytosolic carbon metabolism to thrive in the harsh abiotic conditions typical of intertidal zones.

Unknown to Known: Advancing Knowledge of Coral Gene Function.

Given the catastrophic changes befalling coral reefs, understanding coral gene function is essential to advance reef conservation. This has proved challenging due to the paucity of genomic data and genetic tools available for corals. Recently, CRISPR/Cas9 gene editing was applied to these species; however, a major bottleneck is the identification and prioritization of candidate genes for manipulation. This issue is exacerbated by the many unknown ('dark') coral genes that may play key roles in the stress response. We review the use of gene coexpression networks that incorporate both known and unknown genes to identify targets for reverse genetic analysis. This approach also provides a framework for the annotation of dark genes in established interaction networks to improve our fundamental knowledge of coral gene function.

Group II intron and repeat-rich red algal mitochondrial genomes demonstrate the dynamic recent history of autocatalytic RNAs.

BACKGROUND: Group II introns are mobile genetic elements that can insert at specific target sequences, however, their origins are often challenging to reconstruct because of rapid sequence decay following invasion and spread into different sites. To advance understanding of group II intron spread, we studied the intron-rich mitochondrial genome (mitogenome) in the unicellular red alga, Porphyridium. RESULTS: Analysis of mitogenomes in three closely related species in this genus revealed they were 3-6-fold larger in size (56-132 kbp) than in other red algae, that have genomes of size 21-43 kbp. This discrepancy is explained by two factors, group II intron invasion and expansion of repeated sequences in large intergenic regions. Phylogenetic analysis demonstrates that many mitogenome group II intron families are specific to Porphyridium, whereas others are closely related to sequences in fungi and in the red alga-derived plastids of stramenopiles. Network analysis of intron-encoded proteins (IEPs) shows a clear link between plastid and mitochondrial IEPs in distantly related species, with both groups associated with prokaryotic sequences. CONCLUSION: Our analysis of group II introns in Porphyridium mitogenomes demonstrates the dynamic nature of group II intron evolution, strongly supports the lateral movement of group II introns among diverse eukaryotes, and reveals their ability to proliferate, once integrated in mitochondrial DNA.

Amoeba Genome Reveals Dominant Host Contribution to Plastid Endosymbiosis.

Eukaryotic photosynthetic organelles, plastids, are the powerhouses of many aquatic and terrestrial ecosystems. The canonical plastid in algae and plants originated >1 Ga and therefore offers limited insights into the initial stages of organelle evolution. To address this issue, we focus here on the photosynthetic amoeba Paulinella micropora strain KR01 (hereafter, KR01) that underwent a more recent (∼124 Ma) primary endosymbiosis, resulting in a photosynthetic organelle termed the chromatophore. Analysis of genomic and transcriptomic data resulted in a high-quality draft assembly of size 707 Mb and 32,361 predicted gene models. A total of 291 chromatophore-targeted proteins were predicted in silico, 208 of which comprise the ancestral organelle proteome in photosynthetic Paulinella species with functions, among others, in nucleotide metabolism and oxidative stress response. Gene coexpression analysis identified networks containing known high light stress response genes as well as a variety of genes of unknown function ("dark" genes). We characterized diurnally rhythmic genes in this species and found that over 49% are dark. It was recently hypothesized that large double-stranded DNA viruses may have driven gene transfer to the nucleus in Paulinella and facilitated endosymbiosis. Our analyses do not support this idea, but rather suggest that these viruses in the KR01 and closely related P. micropora MYN1 genomes resulted from a more recent invasion.

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.

Flip-flop organization in the chloroplast genome of Capsosiphon fulvescens (Ulvophyceae, Chlorophyta).

To better understand organelle genome evolution of the ulvophycean green alga Capsosiphon fulvescens, we sequenced and characterized its complete chloroplast genome. The circular chloroplast genome was 111,561 bp in length with 31.3% GC content that contained 108 genes including 77 protein-coding genes, two copies of rRNA operons, and 27 tRNAs. In this analysis, we found the two types of isoform, called heteroplasmy, were likely caused by a flip-flop organization. The flip-flop mechanism may have caused structural variation and gene conversion in the chloroplast genome of C. fulvescens. In a phylogenetic analysis based on all available ulvophycean chloroplast genome data, including a new C. fulvescens genome, we found three major conflicting signals for C. fulvescens and its sister taxon Pseudoneochloris marina within 70 individual genes: (i) monophyly with Ulotrichales, (ii) monophyly with Ulvales, and (iii) monophyly with the clade of Ulotrichales and Ulvales. Although the 70-gene concatenated phylogeny supported monophyly with Ulvales for both species, these complex phylogenetic signals of individual genes need further investigations using a data-rich approach (i.e., organelle genome data) from broader taxon sampling.

Expression of seven carbonic anhydrases in red alga Gracilariopsis chorda and their subcellular localization in a heterologous system, Arabidopsis thaliana.

KEY MESSAGE: Red alga, Gracilariopsis chorda, contains seven carbonic anhydrases that can be grouped into α-, ß- and γ-classes. Carbonic anhydrases (CAHs) are metalloenzymes that catalyze the reversible hydration of CO2. These enzymes are present in all living organisms and play roles in various cellular processes, including photosynthesis. In this study, we identified seven CAH genes (GcCAHs) from the genome sequence of the red alga Gracilariopsis chorda and characterized them at the molecular, cellular and biochemical levels. Based on sequence analysis, these seven isoforms were categorized into four α-class, one ß-class, and two γ-class isoforms. RNA sequencing revealed that of the seven CAHs isoforms, six genes were expressed in G. chorda in light at room temperature. In silico analysis revealed that these seven isoforms localized to multiple subcellular locations such as the ER, mitochondria and cytosol. When expressed as green fluorescent protein fusions in protoplasts of Arabidopsis thaliana leaf cells, these seven isoforms showed multiple localization patterns. The four α-class GcCAHs with an N-terminal hydrophobic leader sequence localized to the ER and two of them were further targeted to the vacuole. GcCAHß1 with no noticeable signal sequence localized to the cytosol. The two γ-class GcCAHs also localized to the cytosol, despite the presence of a predicted presequence. Based on these results, we propose that the red alga G. chorda also employs multiple CAH isoforms for various cellular processes such as photosynthesis.

Parallel evolution of highly conserved plastid genome architecture in red seaweeds and seed plants.

BACKGROUND: The red algae (Rhodophyta) diverged from the green algae and plants (Viridiplantae) over one billion years ago within the kingdom Archaeplastida. These photosynthetic lineages provide an ideal model to study plastid genome reduction in deep time. To this end, we assembled a large dataset of the plastid genomes that were available, including 48 from the red algae (17 complete and three partial genomes produced for this analysis) to elucidate the evolutionary history of these organelles. RESULTS: We found extreme conservation of plastid genome architecture in the major lineages of the multicellular Florideophyceae red algae. Only three minor structural types were detected in this group, which are explained by recombination events of the duplicated rDNA operons. A similar high level of structural conservation (although with different gene content) was found in seed plants. Three major plastid genome architectures were identified in representatives of 46 orders of angiosperms and three orders of gymnosperms. CONCLUSIONS: Our results provide a comprehensive account of plastid gene loss and rearrangement events involving genome architecture within Archaeplastida and lead to one over-arching conclusion: from an ancestral pool of highly rearranged plastid genomes in red and green algae, the aquatic (Florideophyceae) and terrestrial (seed plants) multicellular lineages display high conservation in plastid genome architecture. This phenomenon correlates with, and could be explained by, the independent and widely divergent (separated by >400 million years) origins of complex sexual cycles and reproductive structures that led to the rapid diversification of these lineages.

Taxonomic reinvestigation of the genus Tetradesmus (Scenedesmaceae; Sphaeropleales) based on morphological characteristics and chloroplast genomes.

The genus Tetradesmus (Scenedesmaceae; Sphaeropleales) comprises one of the most abundant green algae in freshwater environments. It includes morphologically diverse species that exhibit bundle-like, plane-arranged coenobia, and unicells, because several different Scenedesmus-like groups were integrated into this genus based on phylogenetic analysis. Nevertheless, there is no clear information regarding the phylogenetic relationship of Tetradesmus species, determined using several marker genes, because of low phylogenetic support and insufficient molecular data. Currently, genome information is available from diverse taxa, which could provide high-resolution evolutionary relationships. In particular, phylogenetic studies using chloroplast genomes demonstrated the potential to establish high-resolution phylogenetic relationships. However, only three chloroplast genomes are available from the genus Tetradesmus. In this study, we newly generated 9 chloroplast genomes from Tetradesmus and constructed a high-resolution phylogeny using a concatenated alignment of 69 chloroplast protein sequences. We also report one novel species (T. lancea), one novel variety (T. obliquus var. spiraformis), and two novel formae (T. dissociatus f. oviformis, T. obliquus f. rectilineare) within the genus Tetradesmus based on morphological characteristics (e.g., cellular arrangements and coenobial types) and genomic features (e.g., different exon-intron structures in chloroplast genomes). Moreover, we taxonomically reinvestigated the genus Tetradesmus based on these results. Altogether, our study can provide a comprehensive understanding of the taxonomic approaches for investigating this genus.

From dusk till dawn: cell cycle progression in the red seaweed Gracilariopsis chorda (Rhodophyta).

The conserved eukaryotic functions of cell cycle genes have primarily been studied using animal/plant models and unicellular algae. Cell cycle progression and its regulatory components in red (Rhodophyta) seaweeds are poorly understood. We analyzed diurnal gene expression data to investigate the cell cycle in the red seaweed Gracilariopsis chorda. We identified cell cycle progression and transitions in G. chorda which are induced by interactions of key regulators such as E2F/DP, RBR, cyclin-dependent kinases, and cyclins from dusk to dawn. However, several typical CDK inhibitor proteins are absent in red seaweeds. Interestingly, the G1-S transition in G. chorda is controlled by delayed transcription of GINS subunit 3. We propose that the delayed S phase entry in this seaweed may have evolved to minimize DNA damage (e.g., due to UV radiation) during replication. Our results provide important insights into cell cycle-associated physiology and its molecular mechanisms in red seaweeds.

Phylogeny and species delimitation of ciliates in the genus Spirostomum (Class, Heterotrichea) using single-cell transcriptomes.

Ciliates are single-celled microbial eukaryotes that diverged from other eukaryotic lineages over a billion years ago. The extensive evolutionary timespan of ciliate has led to enormous genetic and phenotypic changes, contributing significantly to their high level of diversity. Recent analyses based on molecular data have revealed numerous cases of cryptic species complexes in different ciliate lineages, demonstrating the need for a robust approach to delimit species boundaries and elucidate phylogenetic relationships. Heterotrich ciliate species of the genus Spirostomum are abundant in freshwater and brackish environments and are commonly used as biological indicators for assessing water quality. However, some Spirostomum species are difficult to identify due to a lack of distinguishable morphological characteristics, and the existence of cryptic species in this genus remains largely unexplored. Previous phylogenetic studies have focused on only a few loci, namely the ribosomal RNA genes, alpha-tubulin, and mitochondrial CO1. In this study, we obtained single-cell transcriptome of 25 Spirostomum species populations (representing six morphospecies) sampled from South Korea and the USA, and used concatenation- and coalescent-based methods for species tree inference and delimitation. Phylogenomic analysis of 37 Spirostomum populations and 265 protein-coding genes provided a robustious insight into the evolutionary relationships among Spirostomum species and confirmed that species with moniliform and compact macronucleus each form a distinct monophyletic lineage. Furthermore, the multispecies coalescent (MSC) model suggests that there are at least nine cryptic species in the Spirostomum genus, three in S. minus, two in S. ambiguum, S. subtilis, and S. teres each. Overall, our fine sampling of closely related Spirostomum populations and wide scRNA-seq allowed us to demonstrate the hidden crypticity of species within the genus Spirostomum, and to resolve and provide much stronger support than hitherto to the phylogeny of this important ciliate genus.

Metagenome-assembled genomes of the GU0601 sample (the Han River, South Korea).

We generated metagenome sequences of the GU0601 sample collected from the Han River and constructed metagenome-assembled genomes (MAGs) to identify their bacterial composition. We identified six MAGs belonging to Alphaproteobacteria, Cyanobacteria, and Flavobacteria.

Transcriptomic analysis of brine shrimp Artemia franciscana across a wide range of salinities.

Brine shrimp Artemia franciscana, a commercially important species, can thrive in a wide range of salinities and is commonly found in hypersaline lakes and solar salterns. Transcriptome analysis can enhance the understanding of the adaptative mechanisms of brine shrimp in aquaculture. RNA sequencing (RNAseq) data was generated from A. franciscana adults that were salt-adapted for 2-4 weeks at five salinities: 35, 50, 100, 150, and 230 psu. Long-read isoform sequencing (IsoSeq) data was used to construct a high-quality transcriptome assembly. Also, the gene expression patterns in A. franciscana adults were examined. Notably, the transcriptional response of A. franciscana's acclimation to intermediate salinities (50-150 psu) displayed frequently and differentially U-shaped or inverted U-shaped expression patterns. In addition, the types of genes showing two nonmonotonic expression patterns were distinct from each other. The coordinated shifts in gene expression suggest different homeostatic strategies of A. franciscana at specific salinities; such strategies may enhance population fitness at extreme salinities. Our study should promote a scientific concept for the gene expression patterns of A. franciscana along a broad salinity gradient, and a variety of salinity and prey should be monitored for testing the gene expression pattern of this important aquaculture species.

High-quality genome assembles from key Hawaiian coral species.

BACKGROUND: Coral reefs house about 25% of marine biodiversity and are critical for the livelihood of many communities by providing food, tourism revenue, and protection from wave surge. These magnificent ecosystems are under existential threat from anthropogenic climate change. Whereas extensive ecological and physiological studies have addressed coral response to environmental stress, high-quality reference genome data are lacking for many of these species. The latter issue hinders efforts to understand the genetic basis of stress resistance and to design informed coral conservation strategies. RESULTS: We report genome assemblies from 4 key Hawaiian coral species, Montipora capitata, Pocillopora acuta, Pocillopora meandrina, and Porites compressa. These species, or members of these genera, are distributed worldwide and therefore of broad scientific and ecological importance. For M. capitata, an initial assembly was generated from short-read Illumina and long-read PacBio data, which was then scaffolded into 14 putative chromosomes using Omni-C sequencing. For P. acuta, P. meandrina, and P. compressa, high-quality assemblies were generated using short-read Illumina and long-read PacBio data. The P. acuta assembly is from a triploid individual, making it the first reference genome of a nondiploid coral animal. CONCLUSIONS: These assemblies are significant improvements over available data and provide invaluable resources for supporting multiomics studies into coral biology, not just in Hawai'i but also in other regions, where related species exist. The P. acuta assembly provides a platform for studying polyploidy in corals and its role in genome evolution and stress adaptation in these organisms.

Osteochondral autograft transplantation for osteochondritis dissecans of the scaphoid: case report.

This report presents the case of a 36-year-old man who was arthroscopically diagnosed with symptomatic osteochondritis dissecans of the scaphoid. We treated the condition using osteochondral autograft transplantation.

The complete mitochondrial genome of the benthic diatom Pleurosigma inscriptura.

Pleurosigma inscriptura M. A. Harper 2009 is a marine diatom in Naviculales (Bacillariophyceae) order distributed in New Zealand, South America, Argentina, and Korea. We assembled the complete mitochondrial genome sequence of Pleurosigma inscriptura (38,013 bp), and annotated 34 protein-coding genes, 25 transfer RNAs, and 2 ribosomal RNAs. We analyzed a maximum-likelihood tree using conserved 34 mitochondrial genes from Bacillariophyta species. In the mitochondrial phylogeny, P. inscriptura showed a strong monophyletic relationship with Haslea nusantara and Navicula ramosissima.

Percolation threshold of curved linear objects.

In this study, we investigate the percolation threshold of curved linear objects, describing them as quadratic Bézier curves. Using Monte Carlo simulations, we calculate the critical number densities of the curves with different curviness. We also obtain the excluded area of the curves. When an excluded area is given, we can find the critical number density of the curves with arbitrary curviness. Apparent conductivity exponents are computed for the curves, and these values are found to be analogous to that of sticks in the percolative region for a junction resistance dominant system. These results can be used to analyze the optoelectrical performance of metal nanowire films because the high-aspect-ratio metal nanowires can be easily curved during coating.

Complete mitochondrial genome and phylogenetic analysis of the copper shark Carcharhinus brachyurus (Günther, 1870).

Copper shark (Carcharhinus brachyurus Günther, 1870) is one of the most widely distributed but least known species in the family Carcharhinidae. Herein, we report the first complete mitogenome of C. brachyurus. The overall structure of the 16,704 bp C. brachyurus mitogenome was similar to that of other Carcharhinus species and showed the highest average nucleotide identity (97.1%) with the spinner shark (Carcharhinus brevipinna). Multigene phylogeny using 13 protein-coding genes (PCGs) in the mitogenome resolved C. brachyurus clustered with other species within the genus; the overall tree topology was congruent with recent phylogenetic studies of this species. These results provide important information for conservation genetics and further evolutionary studies of sharks.

The complete mitochondrial genome of Pyropia pulchra (Bangiophyceae, Rhodophyta).

Pyropia pulchra (Hollenberg) S.C. Lindstrom & Hughey is a foliose seaweed in Bangiales order distributed in North America. We assembled the complete mitochondrial genome sequence of Pyropia pulchra (33,190 bp), and annotated 26 protein-coding genes, 24 transfer RNAs, and 2 ribosomal RNAs. We analyzed a maximum likelihood tree using conserved 23 mitochondrial genes from Bangiales species. The mitochondrial phylogeny of Bangiales species shows a strong monophyletic relationship of genus Pyropia, and the taxonomic position of P. pulchra within the genus.