Transcriptome and functional analyses of phenotypic plasticity in sea grape Caulerpa okamurae.

Caulerpa is a marine green macroalga distinguished by a large single cell with multiple nuclei. It also exhibits remarkable morphological intraspecies variations, in response to diverse environmental types. However, the molecular mechanisms underlying this phenotypic plasticity remain poorly understood. In this work, we compare the transcriptomes of Caulerpa okamurae Weber Bosse, 1897 displaying altered phenotypes of cultivation and natural phenotypes and investigate significantly regulated genes and their biological functions using differential expression analyses. We observe light-harvesting complex upregulation and cellular framework stability downregulation in altered phenotypes compared to the natural phenotypes. Intertidal macrophytes reduce light capture to avoid photodamage and regulate their morphology to protect against wave damage. In contrast, the lower light conditions and the cultivation environment augment light capture and increase a morphology prioritizing light trapping. Moreover, the addition of simulated wave-sweeping stimuli induces a return to the natural morphology under high-light conditions, showing how mechanical stress affects morphological organization in C. okamurae. We provide detailed gene expression patterns in C. okamurae under varying light intensities and water conditions, suggesting a distinct influence on its morphological traits.

[Effects of heavy metal cadmium on Caulerpa lentillifera based on transcriptome analysis]. / 基于转录组分析重金属镉对长茎葡萄蕨藻的影响.

With the acceleration of industrialization, the toxic effect of heavy metal cadmium (Cd) pollution has become prominent. In order to explore the molecular mechanism of the physiological regulation of Caulerpa lentillifera under Cd stress, we analyzed the transcriptome of Cd-stressed (Hcd2+) algae tissues using RNA-Seq. A total of 702 differentially expressed genes (DEGs) were screened between the control and Hcd2+ groups, out of which 257 genes were up-regulated and 445 genes were down-regulated in the Hcd2+ group. We conducted functional annotation and enrichment analysis of the obtained DEGs. The results showed that various biological functions of C. lentillifera were affected under Cd2+stress, which eventually showed growth inhibition. Results of GO enrichment analysis showed that the production and removal of reactive oxygen species (ROS) in C. lentillifera were out of balance and caused oxidative damage such as DNA damage. Results of KEGG enrichment analysis showed that many photosynthesis-related pathways were inhibited, indicating that Cd2+ stress led to disorder of photosynthetic reaction of C. lentillifera.

Bioinformatic Characterization of Sulfotransferase Provides New Insights for the Exploitation of Sulfated Polysaccharides in Caulerpa.

Caulerpa is an unusual algal genus from Caulerpaceae (Chlorophyta, Bryopsidales). Species from this family produce a wide range of metabolites suitable for biotechnology applications. Among these, sulfated polysaccharides (SPs) are often highly desirable for pharmaceutical and nutraceutical applications. Here, we provide a classification of sulfotransferases from Caulerpa; these important enzymes catalyze the nodal step for the biosynthesis of SPs. For this, we performed phylogenetic, genomic, expression analyses and prediction of the protein structure on sulfotransferases from Caulerpa. Sequences, domains and structures of sulfotransferases generally shared common characteristics with other plants and algae. However, we found an extensive duplication of sulfotransferase gene family, which is unique among the green algae. Expression analysis revealed specific transcript abundance in the pinnae and rachis of the alga. The unique genomic features could be utilized for the production of complex SPs, which require multiple and specific sulfation reactions. The expansion of this gene family in Caulerpaceae would have resulted in a number of proteins characterizing the unique SPs found in these algae. We provide a putative biosynthetic pathway of SPs, indicating the unique characteristics of this pathway in Caulerpa species. These data may help in the future selection of Caulerpa species for both commercial applications and genetic studies to improve the synthesis of valuable products from Caulerpa.

The chloroplast genome sequence of the green macroalga Caulerpa okamurae (Ulvophyceae, Chlorophyta): Its structural features, organization and phylogenetic analysis.

To clarify evolutionary characteristics, phylogenetic relationships as well as species identification of C. okamurae, we determined the cpDNA sequence of Caulerpa okamurae using de novo sequencing in the present study. The cpDNA of C. okamurae was 148,274 bp in length, and it lacked the inverted repeat commonly found in vascular green plants. The cpDNA of C. okamurae was highly compact with a gene density of 71.7%. Moreover, it was an AT-rich genome (65.5%) consisting 76 protein-coding genes (PCGs), 27 transfer RNA (tRNA) genes, three ribosomal RNA (rRNA) genes, 32 putative open reading frames (ORFs) and six introns. Additionally, the six introns were annotated in six genes as follows: psbA, rpoB, ftsH, psbD, atpF and cysA. The overall base composition of its cpDNA was 65.46% for AT. A total of 56 genes were encoded on the light strand, while all the other 50 chloroplast genes were encoded on the heavy strand. All of the PCGs had ATG as their start codon and employed TAA, TGA or TAG as their termination codon. Phylogenetic analyses suggested that the complete cpDNA sequence of C. okamurae fell in the Chlorophyta, Ulvophyceae, Bryopsidales, and Caulerpaceae and more resembled the cpDNAs of C. racemosa, C. cliftonii voucher and Tydemania expeditionis. Taken together, our data offered useful information for the studies of C.okamurae on evolutionary characteristics, phylogenetic relationships as well as species identification.

Differential gene expression in fronds and stolons of the siphonous macroalga, Caulerpa lentillifera.

The green alga, Caulerpa lentillifera, is composed of a single cell with multiple nuclei, but it possesses structures analogous to leaves or fronds, stems or stolons, and roots or rhizoids. To understand molecular mechanisms involved in formation and function of these structures, we carried out RNA-seq analysis of fronds and stolons (including rhizoids). Taking advantage of the decoded genome of C. lentillifera, the present RNA-seq analysis addressed transcripts corresponding to 9,311 genes identified in the genome. RNA-seq data suggested that 8,734 genes are expressed in sporophytes. Despite the siphonous body of the alga, differential gene expression was evident in the two structures. 1,027 (11.8%) and 1,129 (12.9%) genes were preferentially expressed in fronds and stolons, respectively, while the remaining 6,578 (75.3%) genes were expressed at the same level in both. Most genes preferentially expressed in fronds are associated with photosynthesis and plant hormone pathways, including abscisic acid signaling. In contrast, those preferentially expressed in stolons are associated with translation and DNA replication. These results indicate that gene expression is regulated differently between fronds and stolons, which probably governs the function of each structure. Together with genomic information, the present transcriptomic data provide genic information about development and physiology of this unique, siphonous organism.

Promising prospects of nanopore sequencing for algal hologenomics and structural variation discovery.

BACKGROUND: The MinION Access Program (MAP, 2014-2016) allowed selected users to test the prospects of long nanopore reads for diverse organisms and applications through the rapid development of improving chemistries. In 2014, faced with a fragmented Illumina assembly for the chloroplast genome of the green algal holobiont Caulerpa ashmeadii, we applied to the MAP to test the prospects of nanopore reads to investigate such intricacies, as well as further explore the hologenome of this species with native and hybrid approaches. RESULTS: The chloroplast genome could only be resolved as a circular molecule in nanopore assemblies, which also revealed structural variants (i.e. chloroplast polymorphism or heteroplasmy). Signal and Illumina polishing of nanopore-assembled organelle genomes (chloroplast and mitochondrion) reflected the importance of coverage on final quality and current limitations. In hybrid assembly, our modest nanopore data sets showed encouraging results to improve assembly length, contiguity, repeat content, and binning of the larger nuclear and bacterial genomes. Profiling of the holobiont with nanopore or Illumina data unveiled a dominant Rhodospirillaceae (Alphaproteobacteria) species among six putative endosymbionts. While very fragmented, the cumulative hybrid assembly length of C. ashmeadii's nuclear genome reached 24.4 Mbp, including 2.1 Mbp in repeat, ranging closely with GenomeScope's estimate (> 26.3 Mbp, including 4.8 Mbp in repeat). CONCLUSION: Our findings relying on a very modest number of nanopore R9 reads as compared to current output with newer chemistries demonstrate the promising prospects of the technology for the assembly and profiling of an algal hologenome and resolution of structural variation. The discovery of polymorphic 'chlorotypes' in C. ashmeadii, most likely mediated by homing endonucleases and/or retrohoming by reverse transcriptases, represents the first report of chloroplast heteroplasmy in the siphonous green algae. Improving contiguity of C. ashmeadii's nuclear and bacterial genomes will require deeper nanopore sequencing to greatly increase the coverage of these larger genomic compartments.

A siphonous macroalgal genome suggests convergent functions of homeobox genes in algae and land plants.

Genome evolution and development of unicellular, multinucleate macroalgae (siphonous algae) are poorly known, although various multicellular organisms have been studied extensively. To understand macroalgal developmental evolution, we assembled the ∼26 Mb genome of a siphonous green alga, Caulerpa lentillifera, with high contiguity, containing 9,311 protein-coding genes. Molecular phylogeny using 107 nuclear genes indicates that the diversification of the class Ulvophyceae, including C. lentillifera, occurred before the split of the Chlorophyceae and Trebouxiophyceae. Compared with other green algae, the TALE superclass of homeobox genes, which expanded in land plants, shows a series of lineage-specific duplications in this siphonous macroalga. Plant hormone signalling components were also expanded in a lineage-specific manner. Expanded transport regulators, which show spatially different expression, suggest that the structural patterning strategy of a multinucleate cell depends on diversification of nuclear pore proteins. These results not only imply functional convergence of duplicated genes among green plants, but also provide insight into evolutionary roots of green plants. Based on the present results, we propose cellular and molecular mechanisms involved in the structural differentiation in the siphonous alga.

Identification of alternative oxidase encoding genes in Caulerpa cylindracea by de novo RNA-Seq assembly analysis.

Alternative oxidases (AOX) are defined in plants, fungi and algae. The main function of AOX proteins has been described for electron flow through electron transport chain and regulation of mitochondrial retrograde signaling pathway. The roles of AOX proteins have been characterized in reproduction and resistance against oxidative stress, cold stress, starvation, and biotic attacks. Caulerpa cylindracea is an invasive marine green alga. Although the natural habitats of the species are Australia coasts, the impact of the invasion has been monitored through the Mediterranean Sea and the Aegean Sea. C. cylindracea species have advantages against others by showing higher resistance to stress conditions such as cold, starvation, pathogen attacks and by their capability of sexual and vegetative reproduction. Comparing the advantages of C. cylindracea over the niche and defined functional roles of mitochondrial AOX proteins, it is evident that AOX proteins are likely involved in developing those advantageous skills in C. cylindracea. However, there is limited data about biochemical and molecular mechanisms that take part in stress resistance and invasion characteristics. We aimed to identify mitochondrial alternative oxidase encoding genes in C. cylindracea while annotating whole transcriptome data for the species. Samples were collected from Seferihisar/Izmir. Transcriptome analysis from pooled RNA samples revealed 47,400 assembled contigs represented by 33,340 unigenes. Using standalone Blast analysis, we were able to identify two alternative oxidase encoding genes.

Molecular characteristics of polysulfated fractions from the green seaweed Caulerpa cupressoides and actions on thrombin generation in vitro

Caulerpa cupressoides produces sulfated polysaccharides (Cc-SPs) with serpin-dependent anticoagulant effect, but their actions on thrombin generation (TG) are unknown. This study aimed to partially characterize Cc-SPs and examine their potential as modulators of TG. Infrared analysis characterized extract containing three ulvan fractions (Cc-SP1, -SP2 and -SP3) separated by DEAEcellulose chromatography, with differences in the relative proportions of sulfate (10.99-18.38%) and total sugars (46.59-51.12%), without presenting proteins. Charge density patterns and nonSPs varying from 8 to > 100 kDa on agarose and polyacrylamide gel electrophoresis by sequential staining with toluidine blue and stains-all were also confirmed by gel permeation chromatography. The molecular weight of Cc-SP2 was not altered after treatment with 0.4 M HCl up to 5 h. Only Cc-SP2 altered the activated partial thromboplastin time (15 ± 0.3 IU) vs. heparin (193 IU) and abolished at high concentrations (> 4.1 μg) TG by intrinsic pathway in 60-fold diluted human plasma, while at 4.1 μg attenuated TG by 33.87% delaying the lag phase (32 min.) vs. control (28 min.). Cc-SP2 induced concentration-dependent TG in system without cephalin. Heparin abolished TG at 4.15-fold lower amount, but did not stimulate TG. Therefore, Cc-SPs express dual effects on thrombosis in vitro.

The complete mitochondrial genome of the Caulerpa lentillifera (Ulvophyceae, Chlorophyta): Sequence, genome content, organization structure and phylogenetic consideration.

The complete mitochondrial genome is greatly important for studies on genetic structure and phylogenetic relationship at various taxonomic levels. To obtain information about the evolutionary trends of mtDNA in the Ulvophyceae and also to gain insights into the phylogenetic relationships between ulvophytes and other chlorophytes, we determined the mtDNA sequence of Caulerpa lentillifera (sea grape) using de novo mitochondrial genome sequencing. The complete genomic DNA of C. lentillifera was circular and 209,034 bp in length, and it was the largest green-algal mitochondrial genome sequenced to date, with a low gene density of 65.2%, which is reminiscent of the "expanded" pattern of evolution exhibited by embryophyte mtDNAs. The C. lentillifera mtDNA consisted of a typical set of 17 protein-coding genes (PCGs), 20 transfer RNA (tRNA) genes, three ribosomal RNA (rRNA) genes, 42 putative open reading frames (ORFs) and 29 introns, which had homologs in green-algal mtDNAs displaying an "ancestral" or a "reduced-derived" pattern of evolution. The overall base composition of its mitochondrial genome was 24.19% for A, 24.94% for T, 25.80% for G, 25.07% for C and 50.87% for GC. The mitochondrial genome of C. lentillifera was characterized by numerous small intergenic regions and introns, which was clearly different from other green algae. With the exception of the NADH dehydrogenase subunit 6 (ND6), ND1, ATP and three tRNA genes (tRNA-His, tRNA-Thr and tRNA-Ala), all other mitochondrial genes were encoded on the heavy strand. All of the PCGs had ATG as their start codon and employed TAA, TGA or TAG as their termination codon. To gain insights into the evolutionary trends of mtDNA in the Ulvophyceae, we inferred the complete mtDNA sequence of C. lentillifera, an ulvophyte belonging to a distinct, early-diverging lineage. Taken together, our data offered useful information for the studies on phylogenetic hypotheses and phylogenetic relationships of C. lentillifera within the Chlorophyta.

Under the canopy: Community-wide effects of invasive algae in Marine Protected Areas revealed by metabarcoding.

We analysed with multigene (18S and COI) metabarcoding the effects of the proliferation of invasive seaweeds on rocky littoral communities in two Spanish Marine Protected Areas. The invasive algae studied were Caulerpa cylindracea, Lophocladia lallemandii and Asparagopsis armata. They are canopy-forming, landscape-dominant seaweeds, and we were interested in their effects on the underlying communities of meiobenthos and macrobenthos, separated in two size fractions through sieving. A new semiquantitative treatment of metabarcoding data is introduced. The results for both markers showed that the presence of the invasive seaweed had a significant effect on the understory communities for Lophocladia lallemandii and Asparagopsis armata but not for Caulerpa cylindracea. Likewise, changes in MOTU richness and diversity with invasion status varied in magnitude and direction depending on the alga considered. Our results showed that metabarcoding allows monitoring of the less conspicuous, but not least important, effects of the presence of dominant invasive seaweeds.

Potential effects of an invasive seaweed (Caulerpa cylindracea, Sonder) on sedimentary organic matter and microbial metabolic activities.

Caulerpa cylindracea (Sonder), among the most successful marine bio-invaders on a global scale, poses severe threats to biodiversity. However, the effects of this seaweed on the quantity and the biochemical composition of sedimentary organic matter are still poorly known. Since the whole set of sedimentary features affects the availability of substrates for benthic microbial communities, we: i) investigated the biochemical composition of sediments colonized and not-colonized by C. cylindracea, and ii) compared the metabolic patterns of the microbial communities associated with C. cylindracea and in the sediments colonized and not-colonized by the seaweed. Our results show that C. cylindracea can influence the quantity and biochemical composition of sedimentary organic matter (OM), and that microbial populations associated with colonized sediments do have specific metabolic patterns and degradation capacities. Caulerpa cylindracea can also influence the metabolic patterns of the microbial community specifically adapted to degrade compounds released by the seaweed itself, with possible consequences on C cycling.

Entangled fates of holobiont genomes during invasion: nested bacterial and host diversities in Caulerpa taxifolia.

Successful prevention and mitigation of biological invasions requires retracing the initial steps of introduction, as well as understanding key elements enhancing the adaptability of invasive species. We studied the genetic diversity of the green alga Caulerpa taxifolia and its associated bacterial communities in several areas around the world. The striking congruence of α and ß diversity of the algal genome and endophytic communities reveals a tight association, supporting the holobiont concept as best describing the unit of spreading and invasion. Both genomic compartments support the hypotheses of a unique accidental introduction in the Mediterranean and of multiple invasion events in southern Australia. In addition to helping with tracing the origin of invasion, bacterial communities exhibit metabolic functions that can potentially enhance adaptability and competitiveness of the consortium they form with their host. We thus hypothesize that low genetic diversities of both host and symbiont communities may contribute to the recent regression in the Mediterranean, in contrast with the persistence of highly diverse assemblages in southern Australia. This study supports the importance of scaling up from the host to the holobiont for a comprehensive understanding of invasions.

Hologenome theory supported by cooccurrence networks of species-specific bacterial communities in siphonous algae (Caulerpa).

The siphonous algae of the Caulerpa genus harbor internal microbial communities hypothesized to play important roles in development, defense and metabolic activities of the host. Here, we characterize the endophytic bacterial community of four Caulerpa taxa in the Mediterranean Sea, through 16S rRNA amplicon sequencing. Results reveal a striking alpha diversity of the bacterial communities, similar to levels found in sponges and coral holobionts. These comprise (1) a very small core community shared across all hosts (< 1% of the total community), (2) a variable portion (ca. 25%) shared by some Caulerpa taxa but not by all, which might represent environmentally acquired bacteria and (3) a large (>70%) species-specific fraction of the community, forming very specific clusters revealed by modularity in networks of cooccurrence, even in areas where distinct Caulerpa taxa occurred in sympatry. Indirect inferences based on sequence homology suggest that these communities may play an important role in the metabolism of their host, in particular on their ability to grow on anoxic sediment. These findings support the hologenome theory and the need for a holistic framework in ecological and evolutionary studies of these holobionts that frequently become invasive.

An intracellular transcriptomic atlas of the giant coenocyte Caulerpa taxifolia.

Convergent morphologies have arisen in plants multiple times. In non-vascular and vascular land plants, convergent morphology in the form of roots, stems, and leaves arose. The morphology of some green algae includes an anchoring holdfast, stipe, and leaf-like fronds. Such morphology occurs in the absence of multicellularity in the siphonous algae, which are single cells. Morphogenesis is separate from cellular division in the land plants, which although are multicellular, have been argued to exhibit properties similar to single celled organisms. Within the single, macroscopic cell of a siphonous alga, how are transcripts partitioned, and what can this tell us about the development of similar convergent structures in land plants? Here, we present a de novo assembled, intracellular transcriptomic atlas for the giant coenocyte Caulerpa taxifolia. Transcripts show a global, basal-apical pattern of distribution from the holdfast to the frond apex in which transcript identities roughly follow the flow of genetic information in the cell, transcription-to-translation. The analysis of the intersection of transcriptomic atlases of a land plant and Caulerpa suggests the recurrent recruitment of transcript accumulation patterns to organs over large evolutionary distances. Our results not only provide an intracellular atlas of transcript localization, but also demonstrate the contribution of transcript partitioning to morphology, independent from multicellularity, in plants.

Molecular phylogeny and barcoding of Caulerpa (Bryopsidales) based on the tufA, rbcL, 18S rDNA and ITS rDNA genes.

The biodiversity assessment of different taxa of the genus Caulerpa is of interest from the context of morphological plasticity, invasive potential of some species and biotechnological and pharmacological applications. The present study investigated the identification and molecular phylogeny of different species of Caulerpa occurring along the Indian coast inferred from tufA, rbcL, 18S rDNA and ITS rDNA nucleotide sequences. Molecular data confirmed the identification of 10 distinct Caulerpa species: C. veravalensis, C. verticillata, C. racemosa, C. microphysa, C. taxifolia, C. sertularioides, C. scalpelliformis, C. serrulata, C. peltata and C. mexicana. All datasets significantly supported the sister relationship between C. veravalensis and C. racemosa var. cylindracea. It was also concluded from the results that the specimen identified previously as C. microphysa and C. lentillifera could not be considered as separate species. The molecular data revealed the presence of multiple lineages for C. racemosa which can be resolved into separate species. All four markers were used to ascertain their utility for DNA barcoding. The tufA gene proved a better marker with monophyletic association as the main criteria for identification at the species level. The results also support the use of 18S rDNA insertion sequences to delineate the Caulerpa species through character-based barcoding. The ITS rDNA (5.8S-ITS2) phylogenetic analysis also served as another supporting tool. Further, more sequences from additional Caulerpa specimens will need to be analysed in order to support the role of these two markers (ITS rDNA and 18S insertion sequence) in identification of Caulerpa species. The present study revealed the phylogeny of Caulerpa as complete as possible using the currently available data, which is the first comprehensive report illustrating the molecular phylogeny and barcoding of the genus Caulerpa from Indian waters.

Mediterranean species of Caulerpa are polyploid with smaller genomes in the invasive ones.

Caulerpa species are marine green algae, which often act as invasive species with rapid clonal proliferation when growing outside their native biogeographical borders. Despite many publications on the genetics and ecology of Caulerpa species, their life history and ploidy levels are still to be resolved and are the subject of large controversy. While some authors claimed that the thallus found in nature has a haplodiplobiontic life cycle with heteromorphic alternation of generations, other authors claimed a diploid or haploid life cycle with only one generation involved. DAPI-staining with image analysis and microspectrophotometry were used to estimate relative nuclear DNA contents in three species of Caulerpa from the Mediterranean, at individual, population and species levels. Results show that ploidy levels and genome size vary in these three Caulerpa species, with a reduction in genome size for the invasive ones. Caulerpa species in the Mediterranean are polyploids in different life history phases; all sampled C. taxifolia and C. racemosa var. cylindracea were in haplophasic phase, but in C. prolifera, the native species, individuals were found in both diplophasic and haplophasic phases. Different levels of endopolyploidy were found in both C. prolifera and C. racemosa var. cylindracea. Life history is elucidated for the Mediterranean C. prolifera and it is hypothesized that haplophasic dominance in C. racemosa var. cylindracea and C. taxifolia is a beneficial trait for their invasive strategies.