Analysis of diversity and function of epiphytic bacterial communities associated with macrophytes using a metagenomic approach.

Epiphytic bacteria constitute a vital component of aquatic ecosystems, pivotal in regulating elemental cycling. Despite their significance, the diversity and functions of epiphytic bacterial communities adhering to various submerged macrophytes remain largely unexplored. In this study, we employed a metagenomic approach to investigate the diversity and function of epiphytic bacterial communities associated with six submerged macrophytes: Ceratophyllum demersum, Hydrilla verticillata, Myriophyllum verticillatum, Potamogeton lucens, Stuckenia pectinata, and Najas marina. The results revealed that the predominant epiphytic bacterial species for each plant type included Pseudomonas spp., Microbacterium spp., and Stenotrophomonas rhizophila. Multiple comparisons and linear discriminant analysis effect size indicated a significant divergence in the community composition of epiphytic bacteria among the six submerged macrophytes, with 0.3-1% of species uniquely identified. Epiphytic bacterial richness associated with S. pectinata significantly differed from that of both C. demersum and H. verticillata, although no significant differences were observed in diversity and evenness. Functionally, notable variations were observed in the relative abundances of genes associated with carbon, nitrogen, and phosphorus cycling within epiphytic bacterial communities on the submerged macrophyte hosts. Among these communities, H. verticillata exhibited enrichment in genes related to the 3-hydroxypropionate bicycle and nitrogen assimilation, translocation, and denitrification. Conversely, M. verticillatum showcased enrichment in genes linked to the reductive citric acid cycle (Arnon-Buchanan cycle), reductive pentose phosphate cycle (Calvin cycle), polyphosphate degradation, and organic nitrogen metabolism. In summary, our findings offer valuable insights into the diversity and function of epiphytic bacteria on submerged macrophyte leaves, shedding light on their roles in lake ecosystems.

Antioxidant Effects of Rhodoxanthin from Potamogeton crispus L. on H2 O2 -Induced RAW264.7 Macrophages Cells.

Potamogeton crispus L. (P. crispus) is the type of a widely distributed perennial herbs, which is rich in rhodoxanthin. In this research work, five antioxidant indexes in vitro were selected to study the antioxidant activity of rhodoxanthin from P. crispus (RPC). A model of hydrogen peroxide (H2 O2 ) -induced oxidative damage in RAW264.7 cells was established to analyze the antioxidant effect and potential mechanism of RPC. The levels of ROS, MDA and the activities of oxidation related enzymes by H2 O2 were determined by enzyme linked immunosorbent assay (ELISA). The mRNA expression of Nrf-2, HO-1, SOD1 and SOD2 was measured by qRT-PCR assay. According to the results, RPC had free radical scavenging ability for 2, 2-diphenyl-1-trinitrohydrazine (DPPH), 2,2'-azinobis(3-ethylbenzo-thiazoline-6-sulfonic acid radical ion) (ABTS), hydroxyl radical and superoxide anion. RPC significantly decreased the level of MDA and ROS and LDH activity, while increased GSH level and activities of SOD, GSH-Px and CAT. It was showed that RPC could increase the mRNA expression of Nrf-2, HO-1, SOD1 and SOD2 in RAW264.7 cells in a dose-dependently manner. In summary, RPC treatment could effectively attenuate the H2 O2 -induced cell damage rate, and the mechanism is related to the reduction of H2 O2 induced oxidative stress and the activation of Nrf-2 pathway.

Carbohydrates and polyphenolics of extracts from genetically altered plant acts as catalysts for in vitro synthesis of silver nanoparticle.

Eco-friendly biosynthetic approach for silver nanoparticles production using plant extracts is an exciting advancement in bio-nanotechnology and has been successfully attempted in nearly 41 plant species. However, an established model plant system for systematically unraveling the biochemical components required for silver nanoparticles production is lacking. Here we used Arabidopsis thaliana as the model plant for silver nanoparticles biosynthesis in vitro. Employing biochemical, spectroscopic methods, selected mutants and over-expressor plants of Arabidopsis involved in pleotropic functions and sugar homeostasis, we show that carbohydrates, polyphenolics and glyco-proteins are essential components which stimulated silver nanoparticles synthesis. Using molecular genetics as a tool, our data enforces the requirement of sugar conjugated proteins as essentials for AgNPs synthesis over protein alone. Additionally, a comparative analysis of AgNPs synthesis using the aqueous extracts of some of the plant species found in a brackish water ecosystem (Gracilaria, Potamogeton, Enteromorpha and Scendesmus) were explored. Plant extract of Potamogeton showed the highest potential of nanoparticles production comparable to that of Arabidopsis among the species tested. Silver nanoparticles production in the model plant Arabidopsis not only opens up a possibility of using molecular genetics tool to understand the biochemical pathways and components in detail for its synthesis.

The endangered Florida pondweed (Potamogeton floridanus) is a hybrid: Why we need to understand biodiversity thoroughly.

Thorough understanding of biodiversity is a fundamental prerequisite for biological research. A lack of taxonomic knowledge and species misidentifications are particularly critical for conservation. Here we present an example of Potamogeton floridanus, the Florida Pondweed, an endangered taxon endemic to a small area in the Florida panhandle, whose taxonomic status remained controversial for more than a century, and all previous attempts to elucidate its identity have failed. We applied molecular approaches to tackle the origin of the mysterious taxon and supplemented them with morphological and anatomical investigations of both historical herbarium collections and plants recently collected in the type area for a comprehensive taxonomic reassessment. Sequencing of two nuclear ribosomal markers and one chloroplast non-coding spacer resulted in the surprising discovery that P. floridanus is a hybrid of P. pulcher and P. oakesianus, with the former being the maternal parent. The hybrid colony is currently geographically isolated from the distribution range of P. oakesianus. We show that previous molecular analyses have failed to reveal its hybrid identity due to inadequate nuclear DNA sequence editing. This is an example how the uncritical use of automized sequence reads can hamper molecular species identifications and also affect phylogenetic tree construction and interpretation. This unique hybrid taxon, P. ×floridanus, adds another case study to the debate on hybrid protection; consequences for its conservation are discussed.

Identification of Hybrids in Potamogeton: Incongruence between Plastid and ITS Regions Solved by a Novel Barcoding Marker PHYB.

Potamogeton is one of the most difficult groups to clarify in aquatic plants, which has an extensive range of interspecific morphological and ecological diversity. Internal transcribed spacer (ITS) is prevalent for phylogenetic analysis in plants. However, most researches demonstrate that ITS has a high percentage of homoplasy in phylogenetic datasets. In this study, eighteen materials were collected in Potamogeton from China and incongruence was shown between the rbcL and ITS phylogenies. To solve the discrepancy, we employed a novel barcode PHYB to improve resolution and accuracy of the phylogenetic relationships. The PHYB phylogeny successfully resolved the incongruence between the rbcL and ITS phylogenies. In addition, six hybrids were confirmed using PHYB, including P. compressus × P. pusillus, P. octandrus × P. oxyphyllus, P. gramineus × P. lucens, P. distinctus × P. natans, P. distinctus × P. wrightii, and S. pectinata × S. amblyophylla. Whereas, only one hybrid was identified (P. compressus × P. pusillus) by ITS, indicating that ITS homoplasy was present in Potamogeton and ITS was completely homogenized to one parental lineage. Thus, ITS might have limited utility for phylogenetic relationships in Potamogeton. It is recommended that a three-locus combination of chloroplast DNA gene, ITS and PHYB is potential to effectively reveal more robust phylogenetic relationships and species identification.

Strong Genetic Differentiation of Submerged Plant Populations across Mountain Ranges: Evidence from Potamogeton pectinatus in Iran.

Biogeographic barriers for freshwater biota can be effective at various spatial scales. At the largest spatial scale, freshwater organisms can become genetically isolated by their high mountain ranges, vast deserts, and inability to cross oceans. Isolation by distance of aquatic plants is expected to be stronger across than alongside mountain ridges whereas the heterogeneity of habitats among populations and temporary droughts may influence connectivity and hamper dispersal. Suitable aquatic plant habitats became reduced, even for the widespread submerged Potamogeton pectinatus L. (also named Stuckenia pectinata) giving structure to various aquatic habitats. We compared the level of genetic diversity in a heterogeneous series of aquatic habitats across Iran and tested their differentiation over distances and across mountain ranges (Alborz and Zagros) and desert zones (Kavir), with values obtained from temperate region populations. The diversity of aquatic ecosystems across and along large geographic barriers provided a unique ecological situation within Iran. P. pectinatus were considered from thirty-six sites across Iran at direct flight distances ranging from 20 to 1,200 km. Nine microsatellite loci revealed a very high number of alleles over all sites. A PCoA, NJT clustering and STRUCTURE analysis revealed a separate grouping of individuals of southeastern Iranian sites and was confirmed by their different nuclear ITS and cpDNA haplotypes thereby indicating an evolutionary significant unit (ESU). At the level of populations, a positive correlation between allelic differentiation Dest with geographic distance was found. Individual-based STRUCTURE analysis over 36 sites showed 7 genetic clusters. FST and RST values for ten populations reached 0.343 and 0.521, respectively thereby indicating that allele length differences are more important and contain evolutionary information. Overall, higher levels of diversity and a stronger differentiation was revealed among Iranian P. pectinatus than previously observed for temperate European regions, due to regional differences across mountain ranges over long distances.

Loss of heterophylly in aquatic plants: not ABA-mediated stress but exogenous ABA treatment induces stomatal leaves in Potamogeton perfoliatus.

Heterophyllous aquatic plants produce aerial (i.e., floating and terrestrial) and submerged leaves-the latter lack stomata-while homophyllous plants contain only submerged leaves, and cannot survive on land. To identify whether differences in morphogenetic potential and/or physiological stress responses are responsible for variation in phenotypic plasticity between two plants types, responses to abscisic acid (ABA) and salinity stress were compared between the closely related, but ecologically diverse pondweeds, Potamogeton wrightii (heterophyllous) and P. perfoliatus (homophyllous). The ABA-treated (1 or 10 µM) P. wrightii plants exhibited heterophylly and produced leaves with stomata. The obligate submerged P. perfoliatus plants were able to produce stomata on their leaves, but there were no changes to leaf shape, and stomatal production occurred only at a high ABA concentration (10 µM). Under salinity stress conditions, only P. wrightii leaves formed stomata. Additionally, the expression of stress-responsive NCED genes, which encode a key enzyme in ABA biosynthesis, was consistently up-regulated in P. wrightii, but only temporarily in P. perfoliatus. The observed species-specific gene expression patterns may be responsible for the induction or suppression of stomatal production during exposure to salinity stress. These results suggest that the two Potamogeton species have an innate morphogenetic ability to form stomata, but the actual production of stomata depends on ABA-mediated stress responses specific to each species and habitat.

A new delimitation of the Afro-Eurasian plant genus Althenia to include its Australasian relative, Lepilaena (Potamogetonaceae) - Evidence from DNA and morphological data.

Althenia (Potamogetonaceae) is an aquatic plant genus disjunctly distributed in the southern- (South Africa's Cape Floristic Region: CFR) and northern- (Mediterranean Eurasia) hemispheres. This genus and its Australasian relative, Lepilaena, share similar floral characters yet have been treated as different genera or sections of Althenia sensu lato (s.l.) due to the isolated geographic distribution as well as the differences in sex expression, stamen construction, and stigma morphology. The diagnostic characters, however, need reevaluation over the boundaries between the entities. Here we tested the taxonomic delimitation between the entities, assessed synapomorphies for evolutionary lineages, and inferred biogeographic history in a phylogenetic framework. Our results indicated that Lepilaena was resolved as non-monophyletic in both plastid DNA and nuclear PhyC trees and Althenia was nested within it. As Althenia has nomenclatural priority, we propose a new delimitation to recognize Althenia s.l., which can be diagnosed by the female flowers with 3-segmented perianths and male flowers with perianths. The previously used diagnostic characters are either autapomorphies or synapomorphies for small lineages within Althenia s.l., and evolutionary transitions to sessile female flowers and narrow leaves characterize larger clades. Biogeographic analyses suggested a Miocene origin of Althenia s.l. in Australasia and indicated at least one inter- and one intra-specific inter-continental dispersal events among Australasia, Mediterranean Eurasia, and CFR need to be hypothesized to explain the current distribution patterns.

Allopatric divergence of Stuckenia filiformis (Potamogetonaceae) on the Qinghai-Tibet Plateau and its comparative phylogeography with S. pectinata in China.

In the aquatic genus Stuckenia, the wide geographic range of S. pectinata and S. filiformis make them suited for examination of topographic and climatic effects on plant evolution. Using nuclear ITS sequence and ten chloroplast sequences, we conducted comparative phylogeographical analyses to investigate their distribution regions and hybrid zones in China, and compare their phylogeographical patterns and demographical histories. These two species were allopatric in China. S. filiformis occurred only on the Qinghai-Tibet Plateau (QTP), whereas S. pectinata occupied a wide range of habitats. These two species formed hybrid zones on the northeastern edge of QTP. Most of the genetic variance of S. filiformis was between the southern and eastern groups on the QTP, showing a significant phylogeographic structure. The geographical isolations caused by the Nyenchen Tanglha Mountains and the Tanggula Mountains promoted intraspecific diversification of alpine plants on the QTP. This study revealed the lack of phylogeographic structure in S. pectinata, due to the continued gene flow among its distribution regions. The ecological niche modeling showed that the distribution ranges of these two herbaceous species did not contract too much during the glacial period.

Comparative studies of thermotolerance: different modes of heat acclimation between tolerant and intolerant aquatic plants of the genus Potamogeton.

BACKGROUND AND AIMS: Molecular-based studies of thermotolerance have rarely been performed on wild plants, although this trait is critical for summer survival. Here, we focused on thermotolerance and expression of heat shock transcription factor A2 (HSFA2) and its putative target gene (chloroplast-localized small heat shock protein, CP-sHSP) in two allied aquatic species of the genus Potamogeton (pondweeds) that differ in survival on land. METHODS: The degree of thermotolerance was examined using a chlorophyll bioassay to assess heat injury in plants cultivated under non- and heat-acclimation conditions. Potamogeton HSFA2 and CP-sHSP genes were identified and their heat-induction was quantified by real-time PCR. KEY RESULTS: The inhibition of chlorophyll accumulation after heat stress showed that Potamogeton malaianus had a higher basal thermotolerance and developed acquired thermotolerance, whereas Potamogeton perfoliatus was heat sensitive and unable to acquire thermotolerance. We found two duplicated HSFA2 and CP-sHSP genes in each species. These genes were induced by heat shock in P. malaianus, while one HSFA2a gene was not induced in P. perfoliatus. In non-heat-acclimated plants, transcript levels of HSFA2 and CP-sHSP were transiently elevated after heat shock. In heat-acclimated plants, transcripts were continuously induced during sublethal heat shock in P. malaianus, but not in P. perfoliatus. Instead, the minimum threshold temperature for heat induction of the CP-sHSP genes was elevated in P. perfoliatus. CONCLUSIONS: Our comparative study of thermotolerance showed that heat acclimation leads to species-specific changes in heat response. The development of acquired thermotolerance is beneficial for survival at extreme temperatures. However, the loss of acquired thermotolerance and plasticity in the minimum threshold temperature of heat response may be favourable for plants growing in moderate habitats with limited daily and seasonal temperature fluctuations.

Internal dispersal of seeds by waterfowl: effect of seed size on gut passage time and germination patterns.

Long distance dispersal may have important consequences for gene flow and community structure. The dispersal of many plants depends on transport by vertebrate seed dispersers. The shapes of seed shadows produced by vertebrates depend both on movement patterns of the dispersers and on the dynamics and effects of passage through the disperser's gut (i.e. the retention time, survival and germination of ingested seeds). A combination of experiments with captive waterbirds and aquatic plant seeds was used to analyse the following: (a) the effects of inter- and intra-specific variation in seed size and duck species on seed retention time in the gut and (b) the relationship between retention time and the percent germination and germination rates of seeds. Among the three Scirpus species used, those with smaller seeds showed higher survival after ingestion by birds and longer retention times inside their guts than those with larger seeds. For Potamogeton pectinatus, only seeds from the smaller size class (<8 mg) survived ingestion. Retention time affected the percent germination and germination rate of Scirpus seeds but in a manner that varied for the different plant and bird species studied. We recorded both linear and non-linear effects of retention time on percent germination. In addition, germination rate was positively correlated with retention time in Scirpus litoralis but negatively correlated in Scirpus lacustris. Small seed size can favour dispersal over larger distances. However, the effects of retention time on percent germination can modify the seed shadows produced by birds due to higher percent germination of seeds retained for short or intermediate periods. The changes in dispersal quality associated with dispersal distance (which is expected to be positively related to retention time) will affect the probability of seedling establishment over longer distances and, thus, the spatial characteristics of the effective seed shadow.

Molecular adaptation of rbcL in the heterophyllous aquatic plant Potamogeton.

BACKGROUND: Heterophyllous aquatic plants show marked phenotypic plasticity. They adapt to environmental changes by producing different leaf types: submerged, floating and terrestrial leaves. By contrast, homophyllous plants produce only submerged leaves and grow entirely underwater. Heterophylly and submerged homophylly evolved under selective pressure modifying the species-specific optima for photosynthesis, but little is known about the evolutionary outcome of habit. Recent evolutionary analyses suggested that rbcL, a chloroplast gene that encodes a catalytic subunit of RuBisCO, evolves under positive selection in most land plant lineages. To examine the adaptive evolutionary process linked to heterophylly or homophylly, we analyzed positive selection in the rbcL sequences of ecologically diverse aquatic plants, Japanese Potamogeton. PRINCIPAL FINDINGS: Phylogenetic and maximum likelihood analyses of codon substitution models indicated that Potamogeton rbcL has evolved under positive Darwinian selection. The positive selection has operated specifically in heterophyllous lineages but not in homophyllous ones in the branch-site models. This suggests that the selective pressure on this chloroplast gene was higher for heterophyllous lineages than for homophyllous lineages. The replacement of 12 amino acids occurred at structurally important sites in the quaternary structure of RbcL, two of which (residue 225 and 281) were identified as potentially under positive selection. CONCLUSIONS/SIGNIFICANCE: Our analysis did not show an exact relationship between the amino acid replacements and heterophylly or homophylly but revealed that lineage-specific positive selection acted on the Potamogeton rbcL. The contrasting ecological conditions between heterophyllous and homophyllous plants have imposed different selective pressures on the photosynthetic system. The increased amino acid replacement in RbcL may reflect the continuous fine-tuning of RuBisCO under varying ecological conditions.

Characterization of two families of tandem repeated DNA sequences in Potamogeton pectinatus L.

DNA sequences belonging to two families of tandem repeats, PpeRsa1 (362-364 bp in length, 62% A+T residues) and PpeRsa2 (355-359 bp in length, 59% A+T residues), have been isolated from the Potamogeton pectinatus L. genome. The two sequence families do not share significant nucleotide sequence similarity, even if an evolutionary relationship between them could be assumed. The comparison of the cleaving activity of isoschizomeres that are either sensitive or insensitive to methylation of cytosine residues in the target sequence revealed high methylation in both sequence families. The copy number per 1C DNA of PpeRsa1- and PpeRsa2-related sequences is estimated to be 4.92 x 10(4) and 7.96 x 10(4), respectively. Taken together, these sequences account for about 7.5% of the entire genome of P. pectinatus. The chromosomal organization of these sequences was investigated by fluorescent in situ hybridization. PpeRsa1 and PpeRsa2 repeats found related sequences in 52 chromosomes of the P. pectinatus complement (2n = 78). The related sequences were localized around the centromeres and at the chromosome ends in three pairs of chromosomes, while they were found only at the chromosome ends in the remaining pairs. Twenty-six chromosomes did not show any hybridization signal. The hypothesis that the species is a hybrid between a diploid parent and an allotetraploid parent is put forward.

Inherited maternal effects on the drought tolerance of a natural hybrid aquatic plant, Potamogeton anguillanus.

We tested whether maternal effects have led to the adaptive divergence of strains of the natural hybrid Potamogeton anguillanus, whose putative parents show contrastingly divergent ecologies. To examine the correlation between phenotypic characters and maternal types, we conducted drought experiments and DNA typing using nuclear and chloroplast genes. In the field, we investigated the distribution of the maternal type along the depth and the inshore-offshore gradient. Hybrids of P. malaianus mothers (M-hybrids) and those of P. perfoliatus mothers (P-hybrids) could not be distinguished morphologically under submerged conditions, but differed in drought tolerance. M-hybrids and P. malaianus formed more terrestrial shoots and exhibited higher survival than P-hybrids and P. perfoliatus in drought experiments. The distribution survey clarified that M-hybrids were dominant in shallow and inshore areas, whereas they were almost absent in deeper and offshore areas. These results indicate that the natural hybrid P. anguillanus differs in adaptive values depending on the maternal type. Bidirectional hybridization and heritable maternal effects may have played important roles in its phenotypic adaptation to local environmental conditions.

Anoxia-enhanced expression of genes isolated by suppression subtractive hybridization from pondweed (Potamogeton distinctus A. Benn.) turions.

Pondweed (Potamogeton distinctus A. Benn.), a monocot aquatic plant species, has turions, which are overwintering buds forming underground as an asexual reproductive organ. Turions not only survive for more than one month but also elongate under strict anoxia, maintaining high-energy charge by activation of fermentation. We cloned 82 cDNA fragments of genes, that are up-regulated during anoxic growth of pondweed turions, by suppression subtractive hybridization. The transcript levels of 44 genes were confirmed to be higher under anoxia than those in air by both Northern blot analysis and a semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) method. A homology search for their nucleotide sequences revealed that some of them are highly homologous to known sequences of genes from other plants. They included alcohol dehydrogenase, pyruvate decarboxylase (PDC), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), vacuolar H(+)-translocating pyrophosphatase and a plasma membrane intrinsic protein. Time courses of transcript accumulation of some genes under anoxia were different from those in air. The activity of PDC increased under anoxic conditions but the activities of GAPDH and pyrophosphatase remained constant after anoxic treatment. Anoxically up-regulated genes are possibly involved in physiological events to control energy production, pH regulation and cell growth under anoxia. These results suggest that transcriptional regulation of these genes serves as an essential part of survival and growth of pondweed turions under anoxia.

[Genetic diversity and clonal structure of Potamogeton malaianus populations].

By using inter-simple sequence repeat (ISSR) makers, this paper studied the genetic diversity and clonal structure of two Potamogeton malaianus populations growing in different habitats of two lakes in the middle reaches of Yangtze River. With six primers selected, a total of 40 fragments, of which 30 (75.0%) were polymorphic, were obtained from 106 individuals. This species showed a high genetic diversity, with Nei's gene diversity of H = 0.2471 and Shannon' s information index of I = 0.3736. A low degree of population differentiation (G(st) = 0.0861) was found between the two populations, and a very high clonal diversity (D = 0.9917) was detected, according to the analysis based on the polymorphic loci. No common genotypes were found among the individuals from the two populations, showing a great clonal differentiation between them. The spatial distribution pattern of P. malaianus genets showed 'guerilla' architecture, and the clonal distance of the population in lake center (3.0 - 31.5 m) was significantly larger than that in lakeshore (2.4 - 6.7 m).

Expression of sucrose synthase genes involved in enhanced elongation of pondweed (Potamogeton distinctus) turions under anoxia.

BACKGROUND AND AIMS: Overwintering buds (turions) of the monocot aquatic pondweed species (Potamogeton distinctus) are highly tolerant to anoxic stress. Sucrose metabolism accompanied by enhanced activity of sucrose synthase (SuSy) operates actively during anaerobic elongation of pondweed turions. The aim of this study is to isolate SuSy genes from the turions and to investigate their transcriptional changes in response to anoxia and other stimuli. METHODS: SuSy genes were isolated from pondweed turions by PCR methods and transcript levels of SuSy genes were examined in response to anoxia, sugars and plant hormones. In addition, the effects of anoxia on SuSy activity were examined both in the soluble fraction and in the microsomal fraction. KEY RESULTS: cDNAs of two SuSy genes (PdSUS1 and PdSUS2) were cloned from pondweed turions. The levels of PdSUS1 transcripts increased under anoxia but did not with sugar treatments. Anoxia-stimulated elongation of turions was further enhanced by 2,4-dichlorophenoxyacetic acid (2,4-D) and suppressed by treatments with sorbitol, 2-deoxyglucose (2-dGlc) and abscisic acid (ABA). The levels of PdSUS1 transcripts were increased by 2,4-D and decreased by sorbitol under anoxia. The levels of PdSUS2 transcripts were not significantly affected by anoxia and any other treatments. SuSy activity of turions under anoxia was enhanced in the soluble fraction, but not in the microsomal fraction. CONCLUSIONS: Up-regulation of PdSUS1 transcription under anoxia may not be attributed to sugar starvation under anoxia. A positive correlation between stem elongation and the level of PdSUS1 transcripts was observed in turions treated with anoxic conditions, 2,4-D and sorbitol. The increase in SuSy activity in the cytosol may contribute to sugar metabolism and sustain stem elongation under anoxia.

Evolutionary divergence and possible incipient speciation in post-glacial populations of a cosmopolitan aquatic plant.

Habitat configuration is expected to have a major influence on genetic exchange and evolutionary divergence among populations. Aquatic organisms occur in two fundamentally different habitat types, the sea and freshwater lakes, making them excellent models to study the contrasting effects of continuity vs. isolation on genetic divergence. We compared the divergence in post-glacial populations of a cosmopolitan aquatic plant, the pondweed Potamogeton pectinatus that simultaneously occurs in freshwater lakes and coastal marine sites. Relative levels of gene flow were inferred in 12 lake and 14 Baltic Sea populations in northern Germany using nine highly polymorphic microsatellite markers developed for P. pectinatus. We found highly significant isolation-by-distance in both habitat types (P < 0.001). Genetic differentiation increased approximately 2.5-times faster among freshwater populations compared with those from the Baltic Sea. As different levels of genetic drift or population history cannot explain these differences, higher population connectivity in the sea relative to freshwater populations is the most likely source of contrasting evolutionary divergence. These findings are consistent with the notion that freshwater angiosperms are more conducive to allopatric speciation than their life-history counterparts in the sea, the relative species poor seagrasses. Surprisingly, population pairs from different habitat types revealed almost maximal genetic divergence expected for complete reproductive isolation, regardless of their respective geographical distance. Hence, the barrier to gene flow between lake and sea habitat types cannot be due to dispersal limitation. We may thus have identified a case of rapid incipient speciation in post-glacial populations of a widespread aquatic plant.

ent-Labdane glycosides from the aquatic plant Potamogeton lucens and analytical evaluation of the lipophilic extract constituents of various Potamogeton species.

Two new ent-labdane glycosides, one known furano-ent-labdane and a new hydroxylated fatty acid were isolated from the dichloromethane extract of the freshwater aquatic plant Potamogeton lucens. The new compounds were assigned the structures of beta-d-glucopyranosyl-8(17),13-ent-labdadien-16,15-olid-18-oate, 18-beta-d-glucopyranosyloxy-8(17),13-ent-labdadien-16,15-olide and 13(R)-hydroxy-octadeca-(9Z,11E,15Z)-trien-oic acid by spectroscopic means. The algicidal activity of these compounds was tested against Raphidocelis subcapitata. Based on our previous study of Potamogeton pectinatus, other constituents were identified in P. lucens by LC-UV-MS, LC-NMR and GC-MS. The lipophilic extract profiles of both species are presented. Two other species, Potamogeton perfoliatus and P. crispus, were also investigated by analytical comparison of their non-polar extracts. The distribution of ent-labdanes characterized in Potamogeton is summarized.

Population differentiation of Potamogeton pectinatus in the Baltic Sea with reference to waterfowl dispersal.

Forty populations of Potamogeton pectinatus L. were sampled from around the Baltic Sea basin. Analysis of 62 ISSR 'loci' showed that the number of clones per population is very variable but shows a tendency to decrease with latitude. Analysis of molecular variance revealed that, overall, just over half the variability is stored within populations and just under half between them (phi(ST) 0.496). In pairwise comparisons, most populations are significantly differentiated. Genetic distance between populations, as measured by phi(ST), increases with geographical distance. Levels of population differentiation, however, are lower on the southeastern Swedish coast than elsewhere, a reduction correlated with the importance of this area as a staging post for the massive migrations of waterfowl from arctic Russia and western Siberia. Cumulative plots of phi(ST) against geographical distance along this coast suggest that, although it does not prevent significant population differentiation, bird traffic reduces it over distances of 150-200 km.