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Costal eutrophication leads to increased sulfide levels in sediments, which has been identified as a major cause of the global decline in seagrass beds. The seagrass Thalassia hemprichii, a dominant tropical species in the Indo-Pacific, is facing a potential threat from sulfide, which can be easily reduced from sulfate in porewater under the influence of global climate change and eutrophication. However, its metabolic response and tolerance mechanisms to high sulfide remain unclear. Thus, the current study investigated the physiological responses and programmed metabolic networks of T. hemprichii through a three-week mesocosm experiment, integrating physiology, stable isotope, widely targeted metabolomics, transcriptomics, and microbial diversity assessments. High sulfide reduced the sediment microbial diversity, while increased sediment sulfate reduced bacterial abundance and δ 34 S. The exposure to sulfide enhanced root δ 34 S while decreased leaf δ 34 S in T. hemprichii. High sulfide was shown to inhibit photosynthesis via damaging PSII, which further reduced ATP production. In response, abundant up-regulated differentially expressed genes in energy metabolism, especially in oxidative phosphorylation, were activated to compensate high energy requirement. High sulfide also promoted autophagy by overexpressing the genes related to phagocytosis and phagolysosome. Meanwhile, metabolomic profiling revealed that the contents of many primary metabolites, such as carbohydrates and amino acids, were reduced in both leaves and roots, likely to provide more energy and synthesize stress-responsive secondary metabolites. Genes related to nitrate reduction and transportation were up-regulated to promote N uptake for sulfide detoxification. High sulfide levels specifically enhanced thiamine in roots, while increased jasmonic acid and flavonoid levels in leaves. The distinct differences in metabolism between roots and leaves might be related to sulfide levels and the growth-defense trade-off. Collectively, our work highlights the specific mechanisms underlying the response and tolerance of T. hemprichii to high sulfide, providing new insights into seagrass strategies for resisting sulfide., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this manuscript., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Halophila beccarii is one of the oldest two generations of seagrass plants and one of the 10 species of seagrass currently at risk of extinction worldwide. Therefore, how to effectively protect the H. beccarii resources from extinction is a huge challenge. Molecular biology research can provide a scientific basis for species conservation. So far, there has been no detailed analysis of the mitochondrial genome of the genus Halophila., Results: The mitochondrial genome of H. beccarii was assembled into 28 circular chromosomes, ranging in length from 41,738 bp to 104,744 bp, with a total length of 1,964,072 bp and a GC content of 46.71%. It contains 39 genes, including 26 protein coding genes, 10 tRNA genes, and 3 rRNA genes. Repeat sequence analysis and prediction of RNA editing sites revealed a total of 850 dispersed repeats, 1,205 simple repeats, 61 tandem repeats, and 120 RNA editing sites. Analysis of codon usage indicates that codons ending in A/U are preferred. Gene migration between the mitochondrial genome and the chloroplast genome was observed through homologous fragment detection. In addition, Ka/Ks analysis showed that most protein coding genes in the mitochondrial genome experienced negative selection, while only the nad3 gene experienced potential positive selection in most Alismatales. Nucleotide polymorphism analysis revealed variations in each gene, with rpl10 being the most significant. In addition, comparative analysis shows that the GC content is conserved, but there are significant differences in the size and structure of mitochondrial genomes among different species of Alismatales. The phylogenetic analysis based on the mitochondrial genome reflects the exact evolutionary and taxonomic status of H. beccarii., Conclusion: In this study, we sequenced and annotated the mitochondrial genome of H. beccarii, and compared it with the mitochondrial genomes of other plants in Alismatales. Our findings enrich the mitogenome database of seagrass plants and highlight the potential for mitochondrial genes to help decipher plant evolutionary history., (© 2024. The Author(s).)

To understand the genomic characteristics of Arctic plants, we generated 28-44 Gb of short-read sequencing data from 13 Arctic plants collected from the High Arctic Svalbard. We successfully estimated the genome sizes of eight species by using the k-mer-based method (180-894 Mb). Among these plants, the mountain sorrel (Oxyria digyna) and Greenland scurvy grass (Cochlearia groenlandica) had relatively small genome sizes and chromosome numbers. We obtained 45 × and 121 × high-fidelity long-read sequencing data. We assembled their reads into high-quality draft genomes (genome size: 561 and 250 Mb; contig N50 length: 36.9 and 14.8 Mb, respectively), and correspondingly annotated 43,105 and 29,675 genes using ~46 and ~85 million RNA sequencing reads. We identified 765,012 and 88,959 single-nucleotide variants, and 18,082 and 7,698 structural variants (variant size ≥ 50 bp). This study provided high-quality genome assemblies of O. digyna and C. groenlandica, which are valuable resources for the population and molecular genetic studies of these plants., (© 2024. The Author(s).)

High concentrations of copper can pollute coastal waters, primarily from agricultural runoff and mining activities, which can harm marine organisms, including seagrasses. The molecular mechanism of copper toxicity to seagrass currently remains unclear. To determine the response to copper, physiological and multi-omic analyses were conducted to explore the molecular mechanism by which copper affects the global threatened seagrass Halophila beccarii Asch. Excessive copper stress causes oxidative damage and stimulates the activity of the antioxidant enzyme system to remove excess reactive oxygen species (ROS), thereby reducing the damage caused by copper stress. Cu increases the activities of superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6), peroxidase (EC 1.11.1.7), ascorbate peroxidase (EC 1.11.1.11), glutathione peroxidase (EC 1.11.1.9), ascorbate oxidase (EC 1.10.3.3), glutathione reductase (EC 1.6.4.2), and dehydroascorbate reductase (EC 1.8.5.1) and the content of malondialdehyde and reduces the activity of monodehydroascorbate reductase (EC 1.6.5.4). Under copper stress, H. beccarii upregulates the metabolic pathways of steroid biosynthesis and cutin, suberin, and wax biosynthesis, downregulates the metabolic pathways of arginine and proline metabolism and fructose and mannose metabolism; the levels of expression of the ribosome-related genes; upregulates the levels of expression of circadian rhythm-related proteins and downregulates the levels of glutathione metabolism and the proteins related to carbon fixation. This study provides new insights into the response of seagrass to copper stress and reports potential candidate metabolites, genes, and proteins that can be considered as biomarkers to improve the protection and management of seagrass meadows., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Objectives: Ottelia Pers. is in the Hydrocharitaceae family. Species in the genus are aquatic, and China is their centre of origin in Asia. Ottelia alismoides (L.) Pers., which is distributed worldwide, is a distinguishing element in China, while other species of this genus are endemic to China. However, O. alismoides is also considered endangered due to habitat loss and pollution in some Asian countries. Ottelia alismoides is the only submerged macrophyte that contains three carbon dioxide-concentrating mechanisms, i.e. bicarbonate (HCO 3 - ) use, crassulacean acid metabolism and the C4 pathway. In this study, we present its first genome assembly to help illustrate the various carbon metabolism mechanisms and to enable genetic conservation in the future., Data Description: Using DNA and RNA extracted from one O. alismoides leaf, this work produced ∼ 73.4 Gb HiFi reads, ∼ 126.4 Gb whole genome sequencing short reads and ∼ 21.9 Gb RNA-seq reads. The de novo genome assembly was 6,455,939,835 bp in length, with 11,923 scaffolds/contigs and an N50 of 790,733 bp. Genome assembly completeness assessment with Benchmarking Universal Single-Copy Orthologs revealed a score of 94.4%. The repetitive sequence in the assembly was 4,875,817,144 bp (75.5%). A total of 116,176 genes were predicted. The protein sequences were functionally annotated against multiple databases, facilitating comparative genomic analysis., (© 2024. The Author(s).)

The fate of fluoroquinolone antibiotics norfloxacin and ofloxacin were investigated in mesocosmic wetlands, along with their effects on nutrients removal, antibiotic resistance genes (ARGs) and epiphytic microbial communities on Hydrilla verticillate using bionic plants as control groups. Approximately 99% of norfloxacin and ofloxacin were removed from overlaying water, and H. verticillate inhibited fluoroquinolones accumulation in surface sediments compared to bionic plants. Partial least squares path modeling showed that antibiotics significantly inhibited the nutrient removal capacity (0.55) but had no direct effect on plant physiology. Ofloxacin impaired wetland performance more strongly than norfloxacin and more impacted the primary microbial phyla, whereas substrates played the most decisive role on microbial diversities. High antibiotics concentration shifted the most dominant phyla from Proteobacteria to Bacteroidetes and inhibited the Xenobiotics biodegradation function, contributing to the aggravation in wetland performance. Dechloromonas and Pseudomonas were regarded as the key microorganisms for antibiotics degradation. Co-occurrence network analysis excavated that microorganisms degrade antibiotics mainly through co-metabolism, and more complexity and facilitation/reciprocity between microbes attached to submerged plants compared to bionic plants. Furthermore, environmental factors influenced ARGs mainly by altering the community dynamics of differential bacteria. This study offers new insights into antibiotic removal and regulation of ARGs accumulation in wetlands with submerged macrophyte., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The inorganic phosphate-solubilizing bacteria (IPB) in the rhizosphere of Hydrilla verticillata can convert insoluble inorganic phosphorus in the environment into soluble phosphorus that can be directly absorbed and utilized by Hydrilla verticillata. In this research, the roots and rhizosphere sediments of Hydrilla verticillata were collected from high-organic matter urban landscape water. The National Botanical Research Institute's Phosphate growth medium (NBRIP medium) was used to screen for efficient cultivable IPB. The 16S rRNA gene sequence analysis was used to determine the taxonomic affiliation of the strains, and ammonium molybdate spectrophotometry was used to detect the phosphate-solubilizing ability of the strains. The results show that a total of 28 IPB strains with good phosphate-solubilizing effect are obtained from the roots and rhizosphere sediments of Hydrilla verticillata. These IPB strains belong to two phyla, four orders, seven classes, nine families, and nine genera. Among these, Bacillus and Acinetobacter are the dominant genera, and the strains SWIH-7, SWIP-6, SWIP-7, SWIP-13, SWIP-15 and SWIP-16 are potential new species. The IPB strains isolated and screened in this research are rich in diversity, with potential new species and stable phosphate-solubilizing characteristic. These IPB strains are suitable for further development as microbial bacterial agents, which can be applied to promote the recovery of submerged plants in polluted water with high-organic matter, treatment of polluted water and ecological restoration of water., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Li et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Seagrasses are a polyphyletic group of monocotyledonous angiosperms that have evolved to live entirely submerged in marine waters. Thus, these species are ideal for studying plant adaptation to marine environments. Herein, we sequenced the chloroplast (cp) genomes of two seagrass species (Zostera muelleri and Halophila ovalis) and performed a comparative analysis of them with 10 previously published seagrasses, resulting in various novel findings., Results: The cp genomes of the seagrasses ranged in size from 143,877 bp (Zostera marina) to 178,261 bp (Thalassia hemprichii), and also varied in size among different families in the following order: Hydrocharitaceae > Cymodoceaceae > Ruppiaceae > Zosteraceae. The length differences between families were mainly related to the expansion and contraction of the IR region. In addition, we screened out 2,751 simple sequence repeats and 1,757 long repeat sequence types in the cp genome sequences of the 12 seagrass species, ultimately finding seven hot spots in coding regions. Interestingly, we found nine genes with positive selection sites, including two ATP subunit genes (atpA and atpF), three ribosome subunit genes (rps4, rps7, and rpl20), one photosystem subunit gene (psbH), and the ycf2, accD, and rbcL genes. These gene regions may have played critical roles in the adaptation of seagrasses to diverse environments. In addition, phylogenetic analysis strongly supported the division of the 12 seagrass species into four previously recognized major clades. Finally, the divergence time of the seagrasses inferred from the cp genome sequences was generally consistent with previous studies., Conclusions: In this study, we compared chloroplast genomes from 12 seagrass species, covering the main phylogenetic clades. Our findings will provide valuable genetic data for research into the taxonomy, phylogeny, and species evolution of seagrasses., (© 2023. The Author(s).)

Background: Hydrocharis L. and Limnobium Rich. are small aquatic genera, including three and two species, respectively. The taxonomic status, phylogenetic relationships and biogeographical history of these genera have remained unclear, owing to the lack of Central African endemic H. chevalieri from all previous studies. We sequenced and assembled plastomes of all three Hydrocharis species and Limnobium laevigatum to explore the phylogenetic and biogeographical history of these aquatic plants., Results: All four newly generated plastomes were conserved in genome structure, gene content, and gene order. However, they differed in size, the number of repeat sequences, and inverted repeat borders. Our phylogenomic analyses recovered non-monophyletic Hydrocharis. The African species H. chevalieri was fully supported as sister to the rest of the species, and L. laevigatum was nested in Hydrocharis as a sister to H. dubia. Hydrocharis-Limnobium initially diverged from the remaining genera at ca. 53.3 Ma, then began to diversify at ca. 30.9 Ma. The biogeographic analysis suggested that Hydrocharis probably originated in Europe and Central Africa., Conclusion: Based on the phylogenetic results, morphological similarity and small size of the genera, the most reasonable taxonomic solution to the non-monophyly of Hydrocharis is to treat Limnobium as its synonym. The African endemic H. chevalieri is fully supported as a sister to the remaining species. Hydrocharis mainly diversified in the Miocene, during which rapid climate change may have contributed to the speciation and extinctions. The American species of former Limnobium probably dispersed to America through the Bering Land Bridge during the Miocene., (© 2022. The Author(s).)

The genus Halophila shows the highest species diversity within the seagrass genera. Southeast Asian countries where several boundary lines exist were considered as the origin of seagrasses. We hypothesize that the boundary lines, such as Wallace's and Lydekker's Lines, may act as marine geographic barriers to the population structure of Halophila major. Seagrass samples were collected at three islands in Vietnamese waters and analyzed by the molecular maker ITS. These sequences were compared with published ITS sequences from seagrasses collected in the whole region of interest. In this study, we reveal the haplotype and nucleotide diversity, linking population genetics, phylogeography, phylogenetics and estimation of relative divergence times of H. major and other members of the Halophila genus. The morphological characters show variation. The results of the ITS marker analysis reveal smaller groups of H. major from Myanmar, Shoalwater Bay (Australia) and Okinawa (Japan) with high supporting values. The remaining groups including Sri Lanka, Viet Nam, the Philippines, Thailand, Malaysia, Indonesia, Two Peoples Bay (Australia) and Tokushima (Japan) showed low supporting values. The Wallacea region shows the highest haplotype and also nucleotide diversity. Non-significant differences were found among regions, but significant differences were presented among populations. The relative divergence times between some members of section Halophila were estimated 2.15-6.64 Mya., Competing Interests: The authors have declared that no competing interests exist.

The Red Sea is one of the most biodiverse aquatic ecosystems. Notably, seagrasses possess a crucial ecological significance. Among them are the two taxa Halophila stipulacea (Forsk.) Aschers., and Thalassia hemprichii (Ehrenb. ex Solms) Asch., which were formally ranked together with the genus Enhalus in three separate families. Nevertheless, they have been recently classified as three subfamilies within Hydrocharitaceae. The interest of this study is to explore their metabolic profiles through ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS/MS) analysis in synergism with molecular networking and to assess their chemosystematics relationship. A total of 144 metabolites were annotated, encompassing phenolic acids, flavonoids, terpenoids, and lipids. Furthermore, three new phenolic acids; methoxy benzoic acid- O -sulphate ( 16 ), O -caffeoyl- O -hydroxyl dimethoxy benzoyl tartaric acid ( 26 ), dimethoxy benzoic acid- O -sulphate ( 30 ), a new flavanone glycoside; hexahydroxy-monomethoxy flavanone- O -glucoside ( 28 ), and a new steviol glycoside; rebaudioside- O -acetate ( 96 ) were tentatively described. Additionally, the evaluation of the antidiabetic potential of both taxa displayed an inherited higher activity of H. stipulaceae in alleviating the oxidative stress and dyslipidemia associated with diabetes. Hence, the current research significantly suggested Halophila , Thalassia , and Enhalus categorization in three different taxonomic ranks based on their intergeneric and interspecific relationship among them and supported the consideration of seagrasses in natural antidiabetic studies.

Ottelia Pers. is the second largest genus of the family Hydrocharitaceae, including approximately 23 extant species. The genus exhibits a diversity of both bisexual and unisexual flowers, and complex reproductive system comprising cross-pollinated to cleistogamous flowers. Ottelia has been regarded as a pivotal group to study the evolution of Hydrocharitaceae, but the phylogenic relationships and evolutionary history of the genus remain unresolved. Here, we reconstructed a robust phylogenetic framework for Ottelia using 40 newly assembled complete plastomes. Our results resolved Ottelia as a monophyletic genus consisting of two major clades, which correspond to the main two centers of diversity in Asia and Africa. According to the divergence time estimation analysis, the crown group Ottelia began to diversify around 13.09 Ma during the middle Miocene. The biogeographical analysis indicated the existence of the most recent common ancestor somewhere in Africa/Australasia/Asia. Basing on further insights from the morphological evolution of Ottelia, we hypothesized that the ancestral center of origin was in Africa, from where the range expanded by transoceanic dispersal to South America and Australasia, and further from Australasia to Asia. We suggested that the climatic change and global cooling since the mid-Miocene, such as the development of East Asian monsoon climate and tectonic movement of the Yunnan-Guizhou Plateau (YGP), might have played a crucial role in the evolution of Ottelia in China., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Revealing cryptic diversity is of great importance for effective conservation and understanding macroevolution and ecology of plants. Ottelia, a typical example of aquatic plants, possesses extremely variable morphology and the presence of cryptic diversity makes its classification problematic. Previous studies have revealed cryptic Ottelia species in Asia, but very little is known about the molecular systematics of this genus in Africa, a center of species diversity of Ottelia. In this study, we sampled Ottelia ulvifolia, an endemic species of tropical Africa, from Zambia and Cameroon. We used six chloroplast DNA regions, nrITS and six polymorphic microsatellite markers to estimate the molecular diversity and population genetic structure in O. ulvifolia. The phylogenetic inference, STACEY and STRUCTURE analyses supported at least three clusters within O. ulvifolia, each representing unique flower types (i.e., bisexual yellow flower, unisexual yellow flower and bisexual white flower types). Although abundant genetic variation (> 50%) was observed within the populations, excessive anthropogenic activities may result in genetic drift and bottlenecks. Here, three cryptic species of O. ulvifolia complex are defined, and insights are provided into the taxonomy of Ottelia using the phylogenetic species concept.

Biotic resistance mediated by native plant diversity has long been hypothesized to reduce the success of invading plant species in terrestrial systems in temperate regions. However, still little is known about the mechanisms driving invasion patterns in other biomes or latitudes. We help to fill this gap by investigating how native plant community presence and diversity, and the presence of native phylogenetically closely related species to an invader, would affect invader Hydrilla verticillata establishment success in tropical freshwater submerged plant communities. The presence of a native community suppressed the growth of H. verticillata, but did not prevent its colonisation. Invader growth was negatively affected by native plant productivity, but independent of native species richness and phylogenetic relatedness to the invader. Native plant production was not related to native species richness in our study. We show that resistance in these tropical aquatic submerged plant communities is mainly driven by the presence and biomass of a native community independent of native species diversity. Our study illustrates that resistance provided by these tropical freshwater submerged plant communities to invasive species contrasts to resistance described for other ecosystems. This emphasizes the need to include understudied systems when predicting patterns of species invasiveness and ecosystem invasibility across biomes.

Invasive populations of macrophytes are widely distributed and have been successfully introduced and established in freshwater habitats. Hydrilla verticillata was first recorded in 2005 in the Upper Paraná River floodplain and in 2007 at the Itaipu Reservoir (Brazil-Paraguay border, ca. 300 km downstream from its first record). However, its genetic variability within different sites in South America is unknown. We used nucleotide sequences corresponding to the trnL-trnF fragment cpDNA to genetically characterize populations of H. verticillata in different ecosystems of the Upper Paraná River basin. The results indicated an absence of genetic differentiation within and between populations of the basin, and even individuals collected 600 km apart belonged to the same haplotype. Moreover, H. verticillata populations of the Upper Paraná River basin also matched the dioecious biotype haplotype of the Southern United States and Asia. The identification of this single haplotype suggests that one founder genotype was introduced and established successfully in the Upper Paraná River basin, then, as a consequence of vegetative reproduction and the dispersal of propagules, spread to different habitats. However, firm conclusions about this inference can only be obtained with markers of biparental inheritance.

The ability to detect founding populations of invasive species or rare species with low number of individuals is important for aquatic ecosystem management. Traditional approaches use historical data, knowledge of the species' ecology and time-consuming surveys. Within the past decade, environmental DNA (eDNA) has emerged as a powerful additional tracking tool. While much work has been done with animals, comparatively very little has been done with aquatic plants. Here we investigated the transportation and seasonal changes in eDNA concentrations for an invasive aquatic species, Elodea canadensis, in Norway. A specific probe assay was developed using chloroplast DNA to study the fate of the targeted eDNA through space and time. The spatial study used a known source of Elodea canadensis within Lake Nordbytjern 400 m away from the lake outlet flowing into the stream Tveia. The rate of disappearance of E. canadensis eDNA was an order of magnitude loss over about 230 m in the lake and 1550 m in the stream. The time series study was performed monthly from May to October in lake Steinsfjorden harbouring E. canadensis, showing that eDNA concentrations varied by up to three orders of magnitude, peaking during fall. In both studies, the presence of suspended clay or turbidity for some samples did not hamper eDNA analysis. This study shows how efficient eDNA tools may be for tracking aquatic plants in the environment and provides key spatial and temporal information on the fate of eDNA., Competing Interests: The authors have declared that no competing interests exist.

Ottelia, a pantropical genus of aquatic plants belonging to the family Hydrocharitaceae, includes several narrowly distributed taxa in Asia. Although the Asian species have received comparatively more research attention than congeners in other areas, various key taxonomic questions remain unaddressed, especially with regards to apparent cryptic diversity within O. alismoides, a widespread species complex native to Asia, northern Australia and tropical Africa. Here we test taxonomic concepts and evaluate species boundaries using a phylogenetic framework. We sampled five of the seven species of Ottelia in Asia as well as each species endemic to Africa and Australia; multiple samples of O. alismoides were obtained from across Asia. Phylogenetic trees based on five plastid DNA markers and the nuclear ITS region shared almost identical topologies. A Bayesian coalescent method of species delimitation using the multi-locus data set discerned one species in Africa, one in Australia and four in Asia with the highest probability. The results lead us to infer that a population sampled in Thailand represents a hitherto unrecognised cryptic taxon within the widespread species complex, although the apparent lack of unambiguous diagnostic characters currently precludes formal description. Conversely, no molecular evidence for distinguishing O. cordata and O. emersa was obtained, and so the latter is synonymised under the former. Two accessions that exhibit inconsistent positions among our phylogenetic trees may represent cases of chloroplast capture, however incomplete lineage sorting or polyploidy are alternative hypotheses that ought to be tested using other molecular markers.

Seagrasses are marine angiosperms that live fully submerged in the sea. They evolved from land plant ancestors, with multiple species representing at least three independent return-to-the-sea events. This raises the question of whether these marine angiosperms followed the same adaptation pathway to allow them to live and reproduce under the hostile marine conditions. To compare the basis of marine adaptation between seagrass lineages, we generated genomic data for Halophila ovalis and compared this with recently published genomes for two members of Zosteraceae, as well as genomes of five non-marine plant species (Arabidopsis, Oryza sativa, Phoenix dactylifera, Musa acuminata, and Spirodela polyrhiza). Halophila and Zosteraceae represent two independent seagrass lineages separated by around 30 million years. Genes that were lost or conserved in both lineages were identified. All three species lost genes associated with ethylene and terpenoid biosynthesis, and retained genes related to salinity adaptation, such as those for osmoregulation. In contrast, the loss of the NADH dehydrogenase-like complex is unique to H. ovalis. Through comparison of two independent return-to-the-sea events, this study further describes marine adaptation characteristics common to seagrass families, identifies species-specific gene loss, and provides molecular evidence for convergent evolution in seagrass lineages.

Ottelia acuminata is an edible aquatic plant species that is endemic to southwestern China. This plant has experienced habitat degradation resulting from environmental change and extensive human disturbance. Determining the genetic variation and genetic structure of O. acuminata populations could help develop strategies to collect, evaluate, utilize and conserve the species. To this end, we genotyped 183 individuals sampled throughout the species distribution using twelve novel nuclear microsatellite loci (nSSRs). Eight of these nSSRs exhibited low average levels of genetic diversity (H E  = 0.351, Ho = 0.376) and showed evidence of significant inbreeding across several populations. A high degree of genetic differentiation was identified among populations (F ST  = 0.457), probably resulting from limited pollen and seed-mediated gene flow. Only 17.8% of variation existed between O. acuminata var. acuminata and other O. acuminata varieties. Bayesian analysis and a UPGMA dendrogram based on Nei's genetic distance also revealed notably low genetic differentiation among the varieties. This low genetic differentiation is possibly attributed to shared ancestral polymorphisms since their divergence. Additional taxonomic and phylogenetic studies with additional molecular markers are needed to determine the population genetic relationship between O. acuminata varieties. Conservation of this species depends on in situ and ex situ actions, such as controlling habitat water pollution and overexploitation and creating a germplasm bank based on the population genetic differences. To the best of our knowledge, this study represents the first attempt to understand the population genetics of O. acuminata in China using novel nSSR markers developed from transcriptome sequencing and could contribute to the conservation management of this economic plant.

Monocots are remarkably homogeneous in sharing a common trimerous pentacyclic floral Bauplan. A major factor affecting monocot evolution is the unique origin of the clade from basal angiosperms. The origin of the floral Bauplan of monocots remains controversial, as no immediate sister groups with similar structure can be identified among basal angiosperms, and there are several possibilities for an ancestral floral structure, including more complex flowers with higher stamen and carpel numbers, or strongly reduced flowers. Additionally, a stable Bauplan is only established beyond the divergence of Alismatales. Here, we observed the floral development of five members of the three 'petaloid' Alismatales families Butomaceae, Hydrocharitaceae, and Alismataceae. Outer stamen pairs can be recognized in mature flowers of Alismataceae and Butomaceae. Paired stamens always arise independently, and are either shifted opposite the sepals or close to the petals. The position of stamen pairs is related to the early development of the petals. In Butomaceae, the perianth is not differentiated and the development of the inner tepals is not delayed; the larger inner tepals (petals) only permit the initiation of stamens in antesepalous pairs. Alismataceae has delayed petals and the stamens are shifted close to the petals, leading to an association of stamen pairs with petals in so-called stamen-petal complexes. In the studied Hydrocharitaceae species, which have the monocot floral Bauplan, paired stamens are replaced by larger single stamens and the petals are not delayed. These results indicate that the origin of the floral Bauplan, at least in petaloid Alismatales, is closely linked to the position of stamen pairs and the rate of petal development. Although the petaloid Alismatales are not immediately at the base of monocot divergence, the floral evolution inferred from the results should be a key to elucidate the origin of the floral Bauplan of monocots.

Seagrasses are monocotyledons that grow in intertidal and subtidal zones. Seagrass beds are among ecosystems with the highest ecosystem service value. However, seagrass beds are rapidly decline due to anthropogenic disturbances and global climate change. Enhalus acoroides, a monoecious perennial species, is the largest seagrass in stature. It distributes in tropical regions, with Hainan Island as its northern limits. Clonal diversity and genetic structure of E. acoroides populations in Hainan Island were studied to facilitate its conservation and restoration. We used four polymorphic microsatellites to genotype samples collected from four extant populations. We found low clonal and genetic diversities within populations, consistent with the expectations of genetic structure in edge populations. A large range of genetic differentiation was found between these four populations, probably due to the large range of geographic distances between them and genetic drift by local extinction and recolonization. We found no sign of recent bottlenecks in all the populations, probably due to the within-population genetic diversity being too low to show obvious reduction even after bottlenecks. We proposed suggestions on population conservation for those with high priority. Given that seagrass bed had experienced rapid decline in recent years, strengthened conservation and ecological restoration are urgently needed.

Premise of the Study: Pollen dispersal is a key process that influences ecological and evolutionary dynamics of plant populations by facilitating sexual reproduction and gene flow. Habitat loss and fragmentation have the potential to reduce pollen dispersal within and among habitat patches. We assessed aquatic pollen dispersal and mating system characteristics in Vallisneria americana-a water-pollinated plant with a distribution that has been reduced from historic levels., Methods: We examined pollen neighborhood size, biparental inbreeding, and pollen dispersal, based on seed paternity using the indirect paternity method KinDist, from samples of 18-39 mothers and 14-20 progeny per mother from three sites across 2 years., Key Results: On average, fruits contained seeds sired by seven fathers. We found significant biparental inbreeding and limited pollen dispersal distances (0.8-4.34 m). However, in a number of cases, correlated paternity did not decline with distance, and dispersal could not be reliably estimated., Conclusions: Frequent pollen dispersal is not expected among patches, and even within patches, gene flow via pollen will be limited. Limited pollen dispersal establishes genetic neighborhoods, which, unless overcome by seed and propagule dispersal, will lead to genetic differentiation even in a continuous population. Unless loss and fragmentation drive populations to extreme sex bias, local pollen dispersal is likely to be unaffected by habitat loss and fragmentation per se because the spatial scale of patch isolation already exceeds pollen dispersal distances. Therefore, managing specifically for pollen connectivity is only relevant over very short distances., (© 2018 Botanical Society of America.)

Because of the accumulation of heavy metals, Hydrilla verticillata (L.f.) Royle, a rooted submerged perennial aquatic herb, is being developed as a potential tool to clean the aquatic ecosystem polluted by heavy metals. However, its physiological responses for heavy metal remain to be elucidated. Here, through employing proteomics approach, we found that excess Cu significantly induced the expressions of four DNA methylation related proteins in H. verticillata, which were the homologues of two domains rearranged methyltransferases (DRM), a methyltransferases chromomethylase (CMT) and a histone H3 lysine-9 specific SUVH6-like (SUVH6). Consistently, a dramatic change in DNA methylation patterns was detected in excess Cu-exposed H. verticillata. Surprisingly, administration of the NADPH oxidase inhibitors, diphenylene iodonium (DPI) and imidazole (IMZ) that block production of reactive oxygen species (ROS) could trigger the remethylation of genomic sites that were demethylated by excess Cu, indicating that Cu-induced ROS might be another way to affect DNA methylation. Further analysis suggested this changed DNA methylation may be owing to the ROS-induced DNA damage. Taken together, our findings demonstrate that two different ways to influence DNA methylation in excess Cu-treated H. verticillata., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Objective: To investigate the oil body protein and function in seeds of mature seagrass, Thalassia hemprichii., Results: Seeds of mature seagrass T. hemprichii when stained with a fluorescent probe BODIPY showed the presence of oil bodies in intracellular cells. Triacylglycerol was the major lipid class in the seeds. Protein extracted from seagrass seeds was subjected to immunological cross-recognition with land plant seed oil body proteins, such as oleosin and caleosin, resulting in no cross-reactivity. An oleosin-like gene was found in seagrass seeds. Next generation sequencing and sequence alignment indicated that the deduced seagrass seed oleosin-like protein has a central hydrophobic domain responsible for their anchoring onto the surface of oil bodies. Phylogenetic analysis showed that the oleosin-like protein was evolutionarily closer to pollen oleosin than to seed oleosins., Conclusion: Oil body protein found in seagrass seeds represent a distinct class of land seed oil body proteins.

The order Alismatales is a hotspot for evolution of plant mitochondrial genomes characterized by remarkable differences in genome size, substitution rates, RNA editing, retrotranscription, gene loss and intron loss. Here we have sequenced the complete mitogenomes of Zostera marina and Stratiotes aloides, which together with previously sequenced mitogenomes from Butomus and Spirodela, provide new evolutionary evidence of genome size reduction, gene loss and transfer to the nucleus. The Zostera mitogenome includes a large portion of DNA transferred from the plastome, yet it is the smallest known mitogenome from a non-parasitic plant. Using a broad sample of the Alismatales, the evolutionary history of ribosomal protein gene loss is analyzed. In Zostera almost all ribosomal protein genes are lost from the mitogenome, but only some can be found in the nucleus.

Although seagrass species in the genus Halophila are generally distributed in tropical or subtropical regions, H. nipponica has been reported to occur in temperate coastal waters of the northwestern Pacific. Because H. nipponica occurs only in the warm temperate areas influenced by the Kuroshio Current and shows a tropical seasonal growth pattern, such as severely restricted growth in low water temperatures, it was hypothesized that this temperate Halophila species diverged from tropical species in the relatively recent evolutionary past. We used a phylogenetic analysis of internal transcribed spacer (ITS) regions to examine the genetic variability and evolutionary trend of H. nipponica. ITS sequences of H. nipponica from various locations in Korea and Japan were identical or showed very low sequence divergence (less than 3-base pair, bp, difference), confirming that H. nipponica from Japan and Korea are the same species. Halophila species in the section Halophila, which have simple phyllotaxy (a pair of petiolate leaves at the rhizome node), were separated into five well-supported clades by maximum parsimony analysis. H. nipponica grouped with H. okinawensis and H. gaudichaudii from the subtropical regions in the same clade, the latter two species having quite low ITS sequence divergence from H. nipponica (7-15-bp). H. nipponica in Clade I diverged 2.95 ± 1.08 million years ago from species in Clade II, which includes H. ovalis. According to geographical distribution and genetic similarity, H. nipponica appears to have diverged from a tropical species like H. ovalis and adapted to warm temperate environments. The results of divergence time estimates suggest that the temperate H. nipponica is an older species than the subtropical H. okinawensis and H. gaudichaudii and they may have different evolutionary histories.

Understanding spatial patterns of gene flow and genetic structure is essential for the conservation of marine ecosystems. Contemporary ocean currents and historical isolation due to Pleistocene sea level fluctuations have been predicted to influence the genetic structure in marine populations. In the Indo-Australian Archipelago (IAA), the world's hotspot of marine biodiversity, seagrasses are a vital component but population genetic information is very limited. Here, we reconstructed the phylogeography of the seagrass Thalassia hemprichii in the IAA based on single nucleotide polymorphisms (SNPs) and then characterized the genetic structure based on a panel of 16 microsatellite markers. We further examined the relative importance of historical isolation and contemporary ocean currents in driving the patterns of genetic structure. Results from SNPs revealed three population groups: eastern Indonesia, western Indonesia (Sunda Shelf) and Indian Ocean; while the microsatellites supported five population groups (eastern Indonesia, Sunda Shelf, Lesser Sunda, Western Australia and Indian Ocean). Both SNPs and microsatellites showed asymmetrical gene flow among population groups with a trend of southwestward migration from eastern Indonesia. Genetic diversity was generally higher in eastern Indonesia and decreased southwestward. The pattern of genetic structure and connectivity is attributed partly to the Pleistocene sea level fluctuations modified to a smaller level by contemporary ocean currents., (© 2016 John Wiley & Sons Ltd.)

A circadian rhythm of the dinoflagellate Peridinium quadridentatum was studied at a time-series station in the southwestern Gulf of Mexico, in May 2007. Different substrates (water column, the seagrass Thalassia testudinum, macroalgae, coral rubble and sandy sediment surface) were sampled at the site at 1.5-3.5m depth. In the samples of coral rubble, P. quadridentatum was scarce. In the water column, the species showed an abundance peak at 15:00. The cell abundance of P. quadridentatum in Thalassia samples increased from 15:00 until 18:00 (1.81×10(4)cells/gsubstratewet weight), and then continuously decreased until 06:00. Changes in P. quadridentatum cell abundance on macroalgae followed the same trend as on Thalassia, with the maximal value at 18:00. The higher abundance of P. quadridentatum (up to 1.40×10(4)cells/gSWW) in macroalgae samples showed the preference for seaweeds. P. quadridentatum has a neritic tropical-boreal distribution. A new combination is proposed: Peridinium quadridentatum var. trispiniferum., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The environmental DNA (eDNA) method has increasingly been recognized as a powerful tool for monitoring aquatic animal species; however, its application for monitoring aquatic plants is limited. To evaluate eDNA analysis for estimating the distribution of aquatic plants, we compared its estimated distributions with eDNA analysis, visual observation, and past distribution records for the submerged species Hydrilla verticillata. Moreover, we conducted aquarium experiments using H. verticillata and Egeria densa and analyzed the relationships between eDNA concentrations and plant biomass to investigate the potential for biomass estimation. The occurrences estimated by eDNA analysis closely corresponded to past distribution records, and eDNA detections were more frequent than visual observations, indicating that the method is potentially more sensitive. The results of the aquarium experiments showed a positive relationship between plant biomass and eDNA concentration; however, the relationship was not always significant. The eDNA concentration peaked within three days of the start of the experiment in most cases, suggesting that plants do not release constant amounts of DNA. These results showed that eDNA analysis can be used for distribution surveys, and has the potential to estimate the biomass of aquatic plants.

Submersed freshwater macrophytes are considered as relevant indicators for use in bulk bottom sediment contact tests. The purpose of this study was to estimate the validity of endpoints of aquatic plant Elodea canadensis for laboratory genotoxicity testing of natural bottom sediments. The inherent level of chromosome abnormalities (on artificial sediments) in roots of E. canadensis under laboratory conditions was lower than the percentage of abnormal cells in bulk sediments from the Yenisei River. The percentage of abnormal cells in roots of E. canadensis was more sensitive to the presence of genotoxic agents in laboratory contact tests than in the natural population of the plant. The spectra of chromosomal abnormalities that occur in roots of E. canadensis under natural conditions in the Yenisei River and in laboratory contact tests on the bulk bottom sediments from the Yenisei River were similar. Hence, chromosome abnormalities in roots of E. canadensis can be used as a relevant and sensitive genotoxicity endpoint in bottom sediment-contact tests., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Aquatic vascular plants are a distinctive group, differing from terrestrial plants in their growth forms and habitats. Among the various aquatic plant life forms, the evolutionary processes of freshwater submerged species are most likely distinct due to their exclusive occurrence in the discrete and patchy aquatic habitats. Using the chloroplast trnL-F region sequence data, we investigated the phylogeographic structure of a submerged macrophyte, Hydrilla verticillata, the single species in the genus Hydrilla, throughout China, in addition to combined sample data from other countries to reveal the colonisation and diversification processes of this species throughout the world., Results: We sequenced 681 individuals from 123 sampling locations throughout China and identified a significant phylogeographic structure (NST > GST, p < 0.01), in which four distinct lineages occurred in different areas. A high level of genetic differentiation among populations (global FST = 0.820) was detected. The divergence of Hydrilla was estimated to have occurred in the late Miocene, and the diversification of various clades was dated to the Pleistocene epoch. Biogeographic analyses suggested an East Asian origin of Hydrilla and its subsequent dispersal throughout the world., Conclusions: The presence of all four clades in China indicates that China is most likely the centre of Hydrilla genetic diversity. The worldwide distribution of Hydrilla is due to recent vicariance and dispersal events that occurred in different clades during the Pleistocene. Our findings also provide useful information for the management of invasive Hydrilla in North America.

In the outer periclinal cytoplasm of leaf epidermal cells of an aquatic angiosperm Vallisneria, blue light induces "chloroplast de-anchoring", a rapid decline in the resistance of chloroplasts against centrifugal force. Chloroplast de-anchoring is known induced within 1 min of irradiation with high-fluence-rate blue light specifically, preceding the commencement of chloroplasts migration toward the anticlinal cytoplasm. However, its regulatory mechanism has remained elusive, although pharmacological analysis suggested that a calcium release from intracellular calcium stores is necessary for the response. In search of the responsible photoreceptors, immunoblotting analysis using antibodies against phototropins demonstrated that cross-reactive polypeptides of 120-kDa exist in the plasma-membrane fraction prepared from the leaves. In vitro phosphorylation analysis revealed that 120-kDa polypeptides were phosphorylated by exposure to blue light in a fluence-dependent manner. The blue-light-induced phosphorylation activity was sensitive to a Ser/Thr kinase inhibitor, staurosporine, and unusually was retained at a high level for a long time in darkness. Furthermore, phototropin gene homologs (Vallisneria PHOTOTROPIN1 and PHOTOTROPIN2) expressed in leaves were isolated. We propose that calcium-regulated chloroplast de-anchoring, possibly mediated by phototropins, is an initial process of the blue-light-induced avoidance response of chloroplasts in Vallisneria., (© 2014 Institute of Botany, Chinese Academy of Sciences.)

Information on genetic diversity and differentiation of seagrass populations is essential for the conservation of coastal ecosystems. However, little is known about the seagrasses in the Indo-West Pacific Ocean, where the world's highest diversity of seagrasses occurs. The influence of sea currents on these populations is also unknown. We estimated the genetic diversity and population genetic structure and identified reproductive features in Enhalus acoroides populations from the Yaeyama Islands, Hainan Island and the Philippines. The Philippines are situated at the centre of the E. acoroides range, Yaeyama and Hainan are peripheral populations, and the Yaeyama population is at the northern limit of the species range. The powerful Kuroshio Current flows from the Philippines to Yaeyama. Genetic analyses using nine microsatellite markers indicated that reproduction of E. acoroides is mostly sexual. Clonal diversity does not decrease in northern populations, although genetic diversity does. However, the genetic diversity of the Yaeyama populations is greater than that of the Hainan populations. Significant genetic differentiation among most populations was evident; however, the Yaeyama and north-east Philippines populations were genetically similar, despite being separated by ~1100 km. An assignment test suggested that recruitment occurs from the north-east Philippines to Yaeyama. The strong current in this region is probably responsible for the extant genetic diversity and recruitment patterns., (© 2014 John Wiley & Sons Ltd.)

Cytogenetic studies on three species of submerged plants from different parts of the Yenisei river area subjected to radioactive impact of the Krasnoyarsk Mining-and-Chemical Plant and the Electrochemical Factory have been conducted. A high level of irregularities in anatelophase and metaphase of mitoses has been revealed in test samples compared to the control: agglutination and fragmentation of chromosomes, lagging chromosomes, bridges, fragments, misdivisions, and others. The natuie of the disorders indicates that they are related in part to the direct damage to the chromosome structure and in part to damage to the spindle.

Background: The Indo-Pacific region has the largest number of seagrass species worldwide and this region is considered as the origin of the Hydrocharitaceae. Halophila ovalis and its closely-related species belonging to the Hydrocharitaceae are well-known as a complex taxonomic challenge mainly due to their high morphological plasticity. The relationship of genetic differentiation and geographic barriers of H. ovalis radiation was not much studied in this region. Are there misidentifications between H. ovalis and its closely related species? Does any taxonomic uncertainty among different populations of H. ovalis persist? Is there any genetic differentiation among populations in the Western Pacific and the Eastern Indian Ocean, which are separated by the Thai-Malay peninsula? Genetic markers can be used to characterize and identify individuals or species and will be used to answer these questions., Results: Phylogenetic analyses of the nuclear ribosomal internal transcribed spacer region based on materials collected from 17 populations in the Western Pacific and the Eastern Indian Ocean showed that some specimens identified as H. ovalis belonged to the H. major clade, also supported by morphological data. Evolutionary divergence between the two clades is between 0.033 and 0.038, much higher than the evolutionary divergence among H. ovalis populations. Eight haplotypes were found; none of the haplotypes from the Western Pacific is found in India and vice versa. Analysis of genetic diversity based on microsatellite analysis revealed that the genetic diversity in the Western Pacific is higher than in the Eastern Indian Ocean. The unrooted neighbor-joining tree among 14 populations from the Western Pacific and the Eastern Indian Ocean showed six groups. The Mantel test results revealed a significant correlation between genetic and geographic distances among populations. Results from band-based and allele frequency-based approaches from Amplified Fragment Length Polymorphism showed that all samples collected from both sides of the Thai-Malay peninsula were clustered into two clades: Gulf of Thailand and Andaman Sea., Conclusions: Our study documented the new records of H. major for Malaysia and Myanmar. The study also revealed that the Thai-Malay peninsula is a geographic barrier between H. ovalis populations in the Western Pacific and the Eastern Indian Ocean.

Premise of the Study: The discontinuous North American distribution of Najas gracillima has not been explained satisfactorily. Influences of extirpation, nonindigenous introduction, and postglacial migration on its distribution were evaluated using field, fossil, morphological, and molecular data. Najas is a major waterfowl food, and appropriate conservation measures rely on accurate characterization of populations as indigenous or imperiled. •, Methods: Seed lengths of N. gracillima from native Korean populations, a nonindigenous Italian population, and North American populations were compared using digital image analysis. DNA sequence analyses from these regions provided nine nrITS genotypes and eight cpDNA haplotypes. •, Key Results: Najas gracillima seeds from Eurasia and California are shorter than those from eastern North America. Nuclear and chloroplast DNA sequences of N. gracillima from Korea and Italy were identical to California material but differed from native eastern North American plants. Eastern North American specimens of N. gracillima at localities above the last glacial maximum boundary were identical or similar genetically to material from the northeastern United States and Atlantic Coastal Plain and Piedmont but divergent from plants of the Interior Highlands-Mississippi Embayment region. •, Conclusions: In California, N. gracillima is nonindigenous and introduced from Asia. In eastern North America, populations that colonized deglaciated areas were derived primarily from refugia in the Atlantic Coastal Plain and Piedmont. Genetic data indicate initial postglacial migration to northeastern North America, with subsequent westward dispersal into the Upper Great Lakes. These results differentiate potentially invasive California populations from seriously imperiled indigenous eastern North American populations.

This study represents the first report characterizing the biological effects of a lipopolysaccharide (LPS) immune modulator on a marine vascular plant. LPS was shown to serve as a strong elicitor of the early defense response in the subtropical seagrass Thalassia testudinum Banks ex König and was capable of inducing an oxidative burst identified at the single cell level. The formation of reactive oxygen species (ROS), detected by a redox-sensitive fluorescent probe and luminol-based chemiluminescence, included a diphenyleneiodonium sensitive response, suggesting the involvement of an NADPH oxidase. A 900 bp cDNA fragment coding for this enzyme was sequenced and found to encode a NAD binding pocket domain with extensive homology to the Arabidopsis thaliana rbohF (respiratory burst oxidase homolog) gene. The triggered release of ROS occurred at 20 min post-elicitation and was dose-dependent, requiring a minimal threshold of 50 μg/mL LPS. Pharmacological dissection of the early events preceding ROS emission indicated that the signal transduction chain of events involved extracellular alkalinization, G-proteins, phospholipase A2, as well as K(+), Ca(2+), and anion channels. Despite exclusively thriving in a marine environment, seagrasses contain ROS-generating machinery and signal transduction components that appear to be evolutionarily conserved with the well-characterized defense response systems found in terrestrial plants., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Toxic metals polluting aquatic ecosystems are taken up by inhabitants and accumulate in the food web, affecting species at all trophic levels. It is therefore important to have good tools to assess the level of risk represented by toxic metals in the environment. Macrophytes are potential organisms for the identification of metal-responsive biomarkers but are still underrepresented in ecotoxicology. In the present study, we used next-generation sequencing to investigate the transcriptomic response of Elodea nuttallii exposed to enhanced concentrations of Hg and Cd. We de novo assembled more than 60 000 contigs, of which we found 170 to be regulated dose-dependently by Hg and 212 by Cd. Functional analysis showed that these genes were notably related to energy and metal homeostasis. Expression analysis using nCounter of a subset of genes showed that the gene expression pattern was able to assess toxic metal exposure in complex environmental samples and was more sensitive than other end points (e.g., bioaccumulation, photosynthesis, etc.). In conclusion, we demonstrate the feasibility of using gene expression signatures for the assessment of environmental contamination, using an organism without previous genetic information. This is of interest to ecotoxicology in a wider sense given the possibility to develop specific and sensitive bioassays.

Non-indigenous species (NIS) are species living outside their historic or native range. Invasive NIS often cause severe environmental impacts, and may have large economical and social consequences. Elodea (Hydrocharitaceae) is a New World genus with at least five submerged aquatic angiosperm species living in fresh water environments. Our aim was to survey the geographical distribution of cpDNA haplotypes within the native and introduced ranges of invasive aquatic weeds Elodea canadensis and E. nuttallii and to reconstruct the spreading histories of these invasive species. In order to reveal informative chloroplast (cp) genome regions for phylogeographic analyses, we compared the plastid sequences of native and introduced individuals of E. canadensis. In total, we found 235 variable sites (186 SNPs, 47 indels and two inversions) between the two plastid sequences consisting of 112,193 bp and developed primers flanking the most variable genomic areas. These 29 primer pairs were used to compare the level and pattern of intraspecific variation within E. canadensis to interspecific variation between E. canadensis and E. nuttallii. Nine potentially informative primer pairs were used to analyze the phylogeographic structure of both Elodea species, based on 70 E. canadensis and 25 E. nuttallii individuals covering native and introduced distributions. On the whole, the level of variation between the two Elodea species was 53% higher than that within E. canadensis. In our phylogeographic analysis, only a single haplotype was found in the introduced range in both species. These haplotypes H1 (E. canadensis) and A (E. nuttallii) were also widespread in the native range, covering the majority of native populations analyzed. Therefore, we were not able to identify either the geographic origin of the introduced populations or test the hypothesis of single versus multiple introductions. The divergence between E. canadensis haplotypes was surprisingly high, and future research may clarify mechanisms that structure native E. canadensis populations.

Elodea canadensis is an aquatic angiosperm native to North America. It has attracted great attention due to its invasive nature when transported to new areas in its non-native range. We have determined the complete nucleotide sequence of the chloroplast (cp) genome of Elodea. Taxonomically Elodea is a basal monocot, and only few monocot cp genomes representing early lineages of monocots have been sequenced so far. The genome is a circular double-stranded DNA molecule 156,700 bp in length, and has a typical structure with large (LSC 86,194 bp) and small (SSC 17,810 bp) single-copy regions separated by a pair of inverted repeats (IRs 26,348 bp each). The Elodea cp genome contains 113 unique genes and 16 duplicated genes in the IR regions. A comparative analysis showed that the gene order and organization of the Elodea cp genome is almost identical to that of Amborella trichopoda, a basal angiosperm. The structure of IRs in Elodea is unique among monocot species with the whole cp genome sequenced. In Elodea and another monocot Lemna minor the borders between IRs and LSC are located upstream of rps 19 gene and downstream of trnH-GUG gene, while in most monocots, IR has extended to include both trnH and rps 19 genes. A phylogenetic analysis conducted using Bayesian method, based on the DNA sequences of 81 chloroplast genes from 17 monocot taxa provided support for the placement of Elodea together with Lemna as a basal monocot and the next diverging lineage of monocots after Acorales. In comparison with other monocots, the Elodea cp genome has gone through only few rearrangements or gene losses. IR of Elodea has a unique structure among the monocot species studied so far as its structure is similar to that of a basal angiosperm Amborella. This result together with phylogenetic analyses supports the placement of Elodea as a basal monocot to the next diverging lineage of monocots after Acorales. So far, only few cp genomes representing early lineages of monocots have been sequenced and, therefore, this study provides valuable information about the course of evolution in divergence of monocot lineages., (Copyright © 2012 Elsevier B.V. All rights reserved.)

To understand the mechanisms of nitrate uptake by submerged vascular plants, a cDNA for a high-affinity nitrate transporter, NRT2, was isolated from Egeria densa, a submerged monocot. The deduced EdNRT2 protein was similar to the proteins of a conserved NRT2 group in higher plants. Real-time reverse transcription-PCR analysis revealed that after feeding whole plants with 0.2 mM nitrate, the EdNRT2 transcripts were induced in both shoots and roots within 0.5 h, reached the maximum by 1-3 h and then decreased. The EdNRT2 transcript levels in shoots were comparable to those in roots. When nitrate was applied separately to shoots and roots, the EdNRT2 transcripts were induced only in nitrate-treated organs and reached the maximum levels comparable to those in organs when nitrate was applied to whole plants. (15)N-nitrate feeding experiments demonstrated that both shoots and roots are responsible for nitrate uptake and that biomass and (15)N content in shoots was even higher than that in roots. We concluded that EdNRT2 is involved in high-affinity nitrate uptake by shoots and roots of E. densa, that nitrate is taken up independently by shoots and roots and that shoots play an important role in nitrate uptake from aquatic ecosystem.

Premise of the Study: Microsatellite primers were developed in the endangered aquatic herb, Ottelia acuminata, to characterize its genetic diversity and understand its population structure., Methods and Results: Eight polymorphic microsatellite markers were developed from two populations of O. acuminata in China. The number of alleles per locus ranged from one to 15; the observed and expected heterozygosities ranged from 0 to 0.885 and from 0 to 0.888, respectively, in the two populations. Selected loci also amplified successfully in O. sinensis., Conclusions: These microsatellite markers will facilitate further studies on the conservation genetics and evolutionary history of O. acuminata.

Background: Hydrocharitaceae is a fully aquatic monocot family, consists of 18 genera with approximately 120 species. The family includes both fresh and marine aquatics and exhibits great diversity in form and habit including annual and perennial life histories; submersed, partially submersed and floating leaf habits and linear to orbicular leaf shapes. The family has a cosmopolitan distribution and is well represented in the Tertiary fossil record in Europe. At present, the historical biogeography of the family is not well understood and the generic relationships remain controversial. In this study we investigated the phylogeny and biogeography of Hydrocharitaceae by integrating fossils and DNA sequences from eight genes. We also conducted ancestral state reconstruction for three morphological characters., Results: Phylogenetic analyses produced a phylogeny with most branches strongly supported by bootstrap values greater than 95 and Bayesian posterior probability values of 1.0. Stratiotes is the first diverging lineage with the remaining genera in two clades, one clade consists of Lagarosiphon, Ottelia, Blyxa, Apalanthe, Elodea and Egeria; and the other consists of Hydrocharis-Limnobium, Thalassia, Enhalus, Halophila, Najas, Hydrilla, Vallisneria, Nechamandra and Maidenia. Biogeographic analyses (DIVA, Mesquite) and divergence time estimates (BEAST) resolved the most recent common ancestor of Hydrocharitaceae as being in Asia during the Late Cretaceous and Palaeocene (54.7-72.6 Ma). Dispersals (including long-distance dispersal and migrations through Tethys seaway and land bridges) probably played major roles in the intercontinental distribution of this family. Ancestral state reconstruction suggested that in Hydrocharitaceae evolution of dioecy is bidirectional, viz., from dioecy to hermaphroditism, and from hermaphroditism to dioecy, and that the aerial-submerged leaf habit and short-linear leaf shape are the ancestral states., Conclusions: Our study has shed light on the previously controversial generic phylogeny of Hydrocharitaceae. The study has resolved the historical biogeography of this family and supported dispersal as the most likely explanation for the intercontinental distribution. We have also provided valuable information for understanding the evolution of breeding system and leaf phenotype in aquatic monocots.

Premise of the Study: Microsatellite primers were developed for the seagrass Enhalus acoroides to investigate genetic variation and identify clonal structure., Methods and Results: Four polymorphic loci and 32 monomorphic loci were developed in E. acoroides. Two to four alleles per locus were observed at the polymorphic loci across 60 individuals of two E. acoroides populations. The observed and expected heterozygosities within populations ranged from 0.100 to 0.5667 and from 0.0977 to 0.5079, respectively., Conclusions: Our study revealed very low polymorphism in E. acoroides, even at the polymorphic loci. Nevertheless, these primers are a useful tool to study genetic variation, clonal structure, and mating system.

Arsenic (As) is a potential hazard to plants' health, however the mechanisms of its toxicity are yet to be properly understood. To determine the impact of redox state and energetic in stress imposition, plants of Hydrilla verticillata (L.f.) Royle, which are known to be potential accumulator of As, were exposed to 100 and 500 μM arsenate (AsV) for 4 to 96 h. Plants demonstrated significant As accumulation with the maximum being at 500 μM after 96 h (568 μg g(-1) dry weight, dw). The accumulation of As led to a significant increase in the level of reactive oxygen species, nitric oxide, carbonyl, malondialdehyde, and percentage of DNA degradation. In addition, the activity of pro-oxidant enzymes like NADPH oxidase and ascorbate oxidase also showed significant increases. These parameters collectively indicated oxidative stress, which in turn caused an increase in percentage of cell death. These negative effects were seemingly linked to an altered energetic and redox equilibrium [analyzed in terms of ATP/ADP, NADH/NAD, NADPH/NADP, reduced glutathione/oxidized glutathione, and ascorbate/dehydroascobate ratios]. Although there was significant increase in the levels of phytochelatins, the As chelating ligands, a large amount of As was presumably present as free ion particularly at 500 μM AsV, which supposedly produced toxic responses. In conclusion, the study demonstrated that the magnitude of disturbance to redox and energetic equilibrium of plants upon AsV exposure determines the extent of toxicity to plants.

Premise of the Study: Polymorphic microsatellite primers were developed in the vulnerable seagrass Halophila beccarii to investigate genetic variation and provide necessary markers for studying its population genetic structure., Methods and Results: Six polymorphic and six monomorphic microsatellite loci were developed in H. beccarii. Most loci were successfully amplified across 40 H. beccarii individuals collected from three populations from coastal regions of southern China. Two to four alleles per locus were observed at the six polymorphic loci. The highest expected heterozygosity was 0.5737., Conclusions: The results demonstrate low levels of polymorphism in H. beccarii from coastal regions of southern China. They also illustrate that these primers may be useful for studying the mating system and population genetics of H. beccarii on a global scale.

The transfer of C(4) plant traits into C(3) plants has long been a strategy for improving the photosynthetic performance of C(3) plants. The introduction of a pathway mimicking the C(4) photosynthetic pathway into the mesophyll cells of C(3) plants was only a realistic approach when transgenic technology was sufficiently well developed and widely adopted. Here an attempt to introduce a single-cell C(4)-like pathway in which CO(2) capture and release occur in the mesophyll cell, such as the one found in the aquatic plant Hydrilla verticillata (L.f.) Royle, into rice (Oryza sativa L.) is described. Four enzymes involved in this pathway were successfully overproduced in the transgenic rice leaves, and 12 different sets of transgenic rice that overproduce these enzymes independently or in combination were produced and analysed. Although none of these transformants has yet shown dramatic improvements in photosynthesis, these studies nonetheless have important implications for the evolution of C(4) photosynthetic genes and their metabolic regulation, and have shed light on the unique aspects of rice physiology and metabolism. This article summarizes the lessons learned during these attempts to engineer single-cell C(4) rice.

Premise of the Study: Microsatellite primers were characterized in Vallisneria natans, a dominant submerged macrophyte occurring in freshwater bodies of tropical and subtropical zones., Methods and Results: Using the Microsatellite Sequence Enrichment protocol, 16 novel polymorphic codominant loci were developed and characterized in V. natans. In addition to these, six existing microsatellite loci from V. spinulosa were successfully amplified and characterized for V. natans. These primers amplified di- and trinucleotide repeats with 2-7 alleles per locus. Most primers also amplified successfully in V. spinulosa and V. denseserrulata., Conclusions: These results indicate the utility of primers in V. natans for future studies of population genetic structure, as well as their applicability across the genus.

Background and Aims: The successful spread of invasive plants in new environments is often linked to multiple introductions and a diverse gene pool that facilitates local adaptation to variable environmental conditions. For clonal plants, however, phenotypic plasticity may be equally important. Here the primary adaptive strategy in three non-native, clonally reproducing macrophytes (Egeria densa, Elodea canadensis and Lagarosiphon major) in New Zealand freshwaters were examined and an attempt was made to link observed differences in plant morphology to local variation in habitat conditions., Methods: Field populations with a large phenotypic variety were sampled in a range of lakes and streams with different chemical and physical properties. The phenotypic plasticity of the species before and after cultivation was studied in a common garden growth experiment, and the genetic diversity of these same populations was also quantified., Key Results: For all three species, greater variation in plant characteristics was found before they were grown in standardized conditions. Moreover, field populations displayed remarkably little genetic variation and there was little interaction between habitat conditions and plant morphological characteristics., Conclusions: The results indicate that at the current stage of spread into New Zealand, the primary adaptive strategy of these three invasive macrophytes is phenotypic plasticity. However, while limited, the possibility that genetic diversity between populations may facilitate ecotypic differentiation in the future cannot be excluded. These results thus indicate that invasive clonal aquatic plants adapt to new introduced areas by phenotypic plasticity. Inorganic carbon, nitrogen and phosphorous were important in controlling plant size of E. canadensis and L. major, but no other relationships between plant characteristics and habitat conditions were apparent. This implies that within-species differences in plant size can be explained by local nutrient conditions. All together this strongly suggests that invasive clonal aquatic plants adapt to a wide range of habitats in introduced areas by phenotypic plasticity rather than local adaptation.