RAPD analysis of genome polymorphism in the family Lemnaceae

The multilocus RAPD analysis of intergeneric, inter-and intraspecific nuclear genome polymorphism was used for the first time to assess intergeneric, interspecific, and intraspecific polymorphism in Lemnaceae growing on the territory of Russia. The origin of the chosen accessions overlapped with the natural range of duckweeds in Russia. Seventy-five Lemnaceae accessions representing eight species (L. minor, L. gibba, L. turionifera, L. japonica, L. trisulca, L. aequinoctialis, S. polyrhiza, and L. punctata) from three genera (Lemna, Spirodela, and Landoltia), were analyzed. The highest variability levels were revealed in L. minor accessions (0.03–0.20). Species L. trisulca and S. polyrhiza were characterized by values of genetic distance 0.01–0.18 and 0.03–0.16, respectively. The lowest polymorphism levels were detected for L. turionifera (0.01–0.11). The dendrogram based on RAPD data showed that L. aequinoctialis was the most genetically distant species of the genus Lemna. Accessions of species L. turionifera and L. japonica, as well as L. minor and L. gibba, did not form separate species-specific subclusters; rather, they fell into clusters with L. japonica/L. turionifera and L. minor/L. gibba. Accessions of the genera Spirodela and Landoltia formed two separate clusters combined into one group.     

THE DISTRIBUTION AND TAXONOMIC SIGNIFICANCE OF LIGNIN IN THE LEMNACEAE

Lignification characteristics of 14 species of Lemnaceae were studied by the alkaline oxidation technique in an attempt to resolve conflicting reports of the presence of lignin and to provide chemotaxonomic data for supplementing previous systematic treatments. Four species of Spirodela, eight of Lemna, Wolffiella oblonga, and Wolffia microscopica were clonally subcultured, homogenized, exhaustively extracted and the insoluble residue oxidized in alkaline cupric hydroxide. Barley (Hordeum vulgare), Elodea densa and Spirogyra sp. were examined to validate the technique. The benzaldehydes were extracted, separated by thin-layer chromatography, and quantitated spectrophotometrically. Recovery of syringaldehyde, vanillin, and to a lesser extent p-hydroxybenzaldehyde, was accepted as evidence that these plants are lignified. The highest yields of syringaldehyde and vanillin were recovered from S. intermedia, the largest member of the Lemnaceae. Lesser amounts were obtained from S. polyrhiza and S. oligorhiza. Spirodela biperforata yielded no syringaldehyde which substantiates its distinctiveness from the morphologically similar S. polyrhiza. All Lemna species are nerved, yet vanillin (but not syringaldehyde) was recovered only from L. trinervis, L. perpusilla, L. minor, and L. minima. The species L. trisulca, L. gibba, L. obscura, and L. valdiviano yielded only p-hydroxybenzaldehyde. Thus the presence of nerves is not evidence for lignification within the genus Lemna, and the relationships suggested by their gross morphology are not supported by their lignin chemistry. Wolffiella is non-lignified by these criteria. Wolffia microscopica is the smallest flowernig plant and it has no nerves; vanillin was unexpectedly recovered and microscopic examination showed highly lignified anther walls.     

DNA barcoding as a tool for early warning and monitoring alien duckweeds (Lemna sp.pl.): the case of Central Italy

Aquatic habitats are vulnerable to the invasion of alien species, so early warning protocols are necessary for eradication. The presence in Italy of two alien duckweeds in freshwaters has been documented: Lemna minuta, that showed high invasivity, and L. valdiviana, still confined to south Lazio. These two species may be mistaken for each other and for the domestic L. minor and L. gibba due to morphological variation. Here, we assess the applicability of DNA barcoding as a complement to morphological analysis for monitoring the spread of alien Lemna. We chose two chloroplast genome sequences for their ability to discriminate all Lemna species: the 5’ intron of the trnK gene and the matK gene. Among 48 samples of Lemna collected at 20 sites in Central Italy, 20 were identified as L. minor, 19 as L. minuta, five as L. trisulca and four as L. gibba. L. minuta was present at most sampling sites; in particular, at six locations of Lake Trasimeno, eight L. minuta samples were found. We demonstrate that DNA sequence analyses with cost-effective barcoding techniques can effectively support expert efforts in species determination for an early alert system of invasive Lemna species.     

Flowering and Endogenous Levels of Benzoic Acid in Lemna Species

The occurrence of benzoic acid, a flower-inducing factor inLemna species, in L. paucicostata strains 151, 381, 321 andL. gibba G3 was established by several purification steps andfinal use of gas liquid chromatography-selected ion monitoring.Quantitative analyses of benzoic acid were made in non-floweringand flowering Lemna to determine differences in levels. Theendogenous level of benzoic acid was shown to vary dependingon culture conditions, but no positive correlation could befound between the endogenous level of benzoic acid and floweringof Lemna. (Received October 6, 1982; Accepted December 23, 1982)     

Analysis of chloroplast <Emphasis Type="Italic">rpS16</Emphasis> intron sequences in Lemnaceae

Chloroplast rpS16 gene intron sequences were determined and characterized for twenty-five Lemnaceae accessions representing nine duckweed species. For each Lemnaceae species nucleotide substitutions and for Lemna minor, Lemna aequinoctialis, Wolffia arrhiza different indels were detected. Most of indels were found for Wolffia arrhiza and Lemna aequinoctialis. The analyses of intraspecific polymorphism resulted in identification of several gaplotypes in Lemna gibba and Lemna trisulca. Lemnaceae phylogenetic relationship based on rpS16 intron variability data has revealed significant differences between Lemna aequinoctialis and other Lemna species. Genetic distance values corroborated competence of Landoltia punctata separations from Spirodela into an independent generic taxon. The acceptability of rpS16 intron sequences for phylogenetic studies in Lemnaceae was shown.     

Allozymic and morphometric variation inLemna minor (Lemnaceae)

Allozymic and morphometric variation was studied in 28 clones ofLemna minor. This variation was compared with the corresponding variation in four clones ofLemna gibba and four clones ofSpirodela polyrrhiza. A high level of allozymic variation was observed among the clones, despite having been grown under uniform laboratory conditions for several years and despite its quasi-exclusive clonal means of propagation. Based on degree of allozymic similarity,Spirodela polyrrhiza was distinguished from the twoLemna species but the latter species were genetically indistinguishable. Allozymic similarity among clones ofLemna minor was not related to morphometric similarity, nor was it related to the degree of geographic separation or climatic similarity of their sites of origin. The results suggest that allozymic variation among these clones ofLemna minor may be largely neutral and not a consequence of differential selection.     

Genetic characterization and barcoding of taxa in the genus Wolffia Horkel ex Schleid. (Lemnaceae) as revealed by two plastidic markers and amplified fragment length polymorphism (AFLP)

The genus Wolffia of the duckweed family (Lemnaceae) contains the smallest flowering plants. Presently, 11 species are recognized and categorized mainly on the basis of morphology. Because of extreme reduction of structure of all species, molecular methods are especially required for barcoding and identification of species and clones of this genus. We applied AFLP combined with Bayesian analysis of population structure to 66 clones covering all 11 species. Nine clusters were identified: (1) W. angusta and W. microscopica (only one clone), (2) W. arrhiza, (3) W. cylindracea (except one clone that might be a transition form), (4) W. australiana, (5) W. globosa, (6) W. globosa, W. neglecta, and W. borealis, (7) W. brasiliensis, and W. columbiana, (8) W. columbiana, (9) W. elongata. Furthermore, we investigated the sequences of plastidic regions rps16 (54 clones) and rpl16 (55 clones), and identified the following species: W. angusta, W. australiana, W. brasiliensis, W. cylindracea, W. elongata, W. microscopica, and W. neglecta. Wolffia globosa has been separated into two groups by both methods. One group which consists only of clones from North America and East Asia was labelled here “typical W. globosa”. The other group of W. globosa, termed operationally “W. neglecta”, contains also clones of W. neglecta and shows high similarity to W. borealis. None of the methods recognized W. borealis as a distinct species. Although each clone could be characterized individually by AFLP and plastidic sequences, and most species could be bar-coded, the presently available data are not sufficient to identify all taxa of Wolffia.     

The Influence of Nicotinic Acid and Plant Hormones on Flowering in Lemna

Nicotinic acid induces flowering in Lemna paucicostata 151 and381 and Lemna gibba G3 when they are grown in one tenth-strengthM medium under continuous light. For L. paucicostata 151 and381, the simultaneous addition of IAA, GA₃ or ABA to the mediumleads to an inhibition of the flower-inducing effect of nicotinicacid, while zeatin leads to a further stimulation of floweringabove that obtained by nicotinic acid alone. By contrast, inL. gibba G3 all four plant hormones inhibit the nicotinic acid-inducedstimulation of flowering. The effect of nicotinic acid on flowering in all three plantsis strongly daylength dependent when the plants are grown inhalf-strength Hutner's medium. Thus, nicotinic acid causes floweringin L. gibba G3 on continuous light but not on 9L:15D or 10L:14Dregimes. In L. paucicostata 381 nicotinic acid has a small effecton 12L:12D regime, a large effect on a 13L:11D regime and noeffect with daylengths longer than 14 hours, and in L. paucicostata151 nicotinic acid is only effective on daylengths shorter thanabout 11 hours. However, in L. paucicostata 151 and 381 treatmentwith both nicotinic acid and zeatin results in flowering undercontinuous light on half-strength Hutner's medium. Nicotinic acid is present in different Lemna but its concentrationdoes not appear to be influenced by changes in daylength. Thus,flowering clearly cannot be controlled by nicotinic acid actingalone, but the results of this study indicate that nicotinicacid could interact with other factors, possibly including oneor more of the known plant hormones, to influence the floweringprocess in Lemna. (Received August 28, 1985; Accepted October 29, 1985)     

AFLP-based differentiation of tropical African Festuca species compared to the European Festuca complex

For the first time amplified fragment length polymorphism (AFLP) fingerprinting is applied to classify tropical African Festuca species. Five afro-alpine narrow- and two afro-montane broad-leaved species from Uganda and Ethiopia are compared to ten European grass species. A principal coordinate analysis (PCoA) accounts for 62.5% with its first three coordinates. The PCoA and the neighbor-joining (NJ) distinguish the five narrow-leaved African Festuca species from all other species. The broad-leaved African Festuca africana and Festuca simensis are linked to the broad-leaved European species through Festuca altissima and Festuca gigantea, respectively. The narrow- and broad-leaved European species are separated as expected in the NJ. One narrow-leaved African alpine species recently described appears merged (i.e. Festuca richardii with Festuca abyssinica). We provide chromosome numbers for all seven Ugandan species and compare taxonomy and AFLP classification. Our most striking result is that the narrow-leaved African Festuca species are unique and not clustering with the narrow-leaved European species.     

Flowering and Endogenous Levels of Plant Hormones in Lemna Species

The occurrence and endogenous level of various plant hormoneswere measured for the short-day plants Lemna paucicostata 151and 381 and the long-day plant Lemna gibba G3 to determine whetherany of them are involved in the photoperiodic control of flowering.ABA, IAA, GA₁, GA₂₉, GA₃₄, GA₅₃, trans- and cis-zeatin, trans-and cis-ribosyl zeatin, N⁶-(²-isopentenyl) adenine and N⁶-(²-isopentenyl)adenosine were definitely detected in each species, while GA₄was only detected in L. gibba G3 and GA₂₀ was only detectedin L. paucicostata 151. The endogenous levels of ABA and IAAwere in the range of 1–7 ng/g fr wt and were not significantlydifferent in vegetative and flowering plants. The endogenousgibberellin levels were generally higher in Lemna grown underlong-day rather than short-day conditions. The endogenous cytokininlevels were almost the same in both flowering and vegetativeplants of L. paucicostata 151 and 381. In L. gibba G3, however,the level of cis-ribosyl zeatin, N⁶-(²-isopentenyl) adenineand N⁶-(²-sopentenyl) adenosine were higher in vegetative thanin flowering plants. These results indicate that there is not necessarily a directrelation between endogenous plant hormone levels and flowering,and that the chemical basis for the photoperiodic control offlowering cannot be explained solely by changes in hormone levels.The possibility remains, however, that one or more of the planthormones has some influence of secondary importance on the floweringprocess in Lemna. (Received January 29, 1986; Accepted July 12, 1986)     

Flower-Inducing Activity of Water Extract of Lemna

Flowering of Lemna paucicostata 441 (P441), a sensitive short-dayplant (SDP), was promoted under a near critical photoperiodby the crude water extract of the same plant added to the medium.The extract induced flowering in L. paucicostata 151 (P151),a weakly responsive SDP, under continuous light. The activityfor P151 was greatly promoted by simultaneous application ofbenzyladenine, and the extract of only 0.3 mg fr wt plant addedto 10 ml of assay medium with 1 µM benzyladenine was active.Active substance(s) was similarly obtained from both flower-inducedand non-induced plants, and more or less from all species andstrains of Lemna tested, including P151. However, the extractof short-day strains was more active than that of L. gibba G3(G3), a long-day strain. G3 responded only slightly to the extractof either P441 or G3, whereas P151 responded far more stronglyto the extract of P441 than to that of G3. (Received April 17, 1989; Accepted August 10, 1989)     

Growth Cycles in Lemna gibba Cultures and Their Effects on Growth Rate and Ultrastructure

A long-established axenic culture of Lemna gibba G3 was maintained in exponential growth phase under controlled conditions. Weekly analyses for 2 years showed that the individual plants of the Lemna gibba clone fluctuated between two forms. One extreme consisted of plants light in weight, small in size, and with a high relative growth rate (RGR), the other of heavy, large, and more slowly growing plants. At intervals plants having intermediate characteristics dominated in the stock culture. Indication of an annual growth-cycle was also found. The magnitude of growth response (weight, RGR, area, and dry matter content) after treatment with abscisic acid (ABA), 6-benzylaminopurine (BAP), and a combination of the two was different for low-weight and heavy plants. The heavy plants were more sensitive to ABA and BAP treatment than the low-weight ones. The accumulation of starch was least in small untreated plants and greatest in ABA treated plants. Large electron transparent globules were found in the chloroplasts of the ABA treated plants and in heavy plants regardless of how they had been treated. The different physiological and ultrastructural characteristics of the two forms of Lemna plants probably reflect an ageing-rejuvenation cycle. Emphasis is placed on the importance of this cycle when Lemna is used as a model plant in physiological experiments.     

Allozyme variation within and divergence between Lemna gibba and L. disperma: Systematic and biogeographic implications

Enzyme electrophoresis was employed to assess genetic diversity within and divergence between Lemna disperma and Lemna gibba, sister species that have often been considered conspecific because of the few technical morphological characters distinguishing them. L. gibba is distributed widely except in Australia and New Zealand, where it is replaced by L. disperma. Allozyme data were employed to examine: (1) whether species recognition is supported by genetic divergence between accessions assigned to the two taxa, (2) whether the level of diversity in the two species supports the hypothesis that L. gibba–L. disperma are related as a progenitor–derivative species pair, and (3) whether estimates of divergence times obtained from allozymes are in general agreement with those from plastid sequences. Accessions of the two species are highly divergent at allozyme loci, with a genetic identity of 0.404, and the putative derivative species (L. disperma) has only one-third the diversity of its proposed ancestor, L. gibba. Therefore, allozyme data support the continued recognition of the two species and are concordant with the hypothesis that the species are related as progenitor and derivative. The reduced morphology of L. disperma and the allozyme data indicate that this species originated via dispersal of L. gibba or of a common ancestor of the two species. Estimated divergence times from allozymes and plastid sequences vary widely, but assuming that actual divergence was within the broad range of estimates, long distance dispersal is required to explain the present distribution of the two species.     

Short term exposure of Lemna minor and Lemna gibba to mercury, cadmium and chromium

The effects of mercury (Hg), cadmium (Cd) and chromium (Cr) in concentrations ranging from 0.02 to 20 mg L^?1 applied for 24 h were assessed in Lemna minor and Lemna gibba by measuring changes in protein concentration, ascorbic acid, phenolics, malondialdehyde (MDA), hydrogen peroxide (H₂O₂), the activity of guaiacol peroxidase (G-POX) and catalase (CAT). Ascorbic acid, phenolics, catalase and guaiacol peroxidase played a key role in the antioxidative response of L. gibba. Inadequate activity of antioxidant enzymes in the L. minor resulted in MDA and H₂O₂ accumulation. In both used species, Hg treatment decreased protein content and increased CAT and G-POX activity, but decreased MDA and H₂O₂ levels. Cadmium and chromium had opposite impacts on two used Lemna species on almost all observed parameters. Enhanced antioxidative responses of L. gibba to lower concentrations of Hg, Cd and Cr indicated greater abiotic stress tolerance than L. minor.     

Out of Africa: molecular phylogenetics and biogeography of Wolffiella (Lemnaceae)

The monophyletic genus Wolffiella (Lemnaceae) comprises 10 species divided taxonomically into three sections. Relative to other genera of Lemnaceae, Wolffiella has a restricted range, with species distributed in warm temperate to tropical areas of Africa and the Americas, with only one species occurring in both areas. Sequence data from coding (rbcL and matK) and non‐coding (trnK and rpl16 introns) regions of cpDNA were analyzed phylogenetically to resolve relationships within Wolffiella, and these results were compared to earlier allozyme and morphological studies. Allozymes, cpDNA and morphology all supported the recognition of three sections. Relationships among species were similar in most respects between the allozyme and cpDNA trees, as well as among the different plastid partitions. In Wolffiella, both non‐synonymous and synonymous substitutions were greater in matK than in rbcL, as observed in other taxa. The synonymous substitution rate in matK was similar to the substitution rate of the non‐coding regions. All partitions, including coding regions, exhibited some homoplasy. Biogeographical reconstructions from a combination of cpDNA partitions indicated that Wolffiella originated in Africa with early movement to and radiation in the Americas. The one species found in both Africa and the Americas, W. welwitschii, likely originated in the Americas and subsequently dispersed to Africa. Using the SOWH test, the cpDNA data could reject two alternative biogeographical hypotheses suggested from analyses of morphological and allozyme data. The present distribution of Wolffiella can be explained by two major dispersal events and this contrasts with the more complex species distributions in other Lemnaceae genera. Limited dispersal in Wolffiella relative to other Lemnaceae genera may be due to more recent origins of species, lower dispersibility and poorer colonizing ability. © 2003 The Linnean Society of London, Biological Journal of the Linnean Society, 2003, 79 , 565–576.     

Genetic structure of duckweed population of Spirodela,Landoltia and Lemna from Lake Tai,China

Main conclusion

Presenting a basic framework for using MLST to characterize Spirodela, Landoltia and in particular Lemna strains at the species level, and to study population genetics and evolution history of natural duckweed populations.

Abstract

Duckweed is widely used in environmental biotechnology and has recently emerged as a potential feedstock for biofuels due to its high growth rate and starch content. The genetic diversity and composition of a natural duckweed population in genera Spirodela, Landoltia and Lemna from Lake Tai, China, were investigated using probabilistic analysis of multilocus sequence typing (MLST). The 78 strains were categorized into five lineages, among which strains representing L. aequinoctialis and S. polyrhiza were predominant. Among the five lineages, interlineage transfers of markers were infrequent and no recombination was statistically detected. Tajima’s D tests determined that all loci are subject to population bottlenecks, which is likely one of the main reasons for the low genetic diversity observed within the lineages. Interestingly, strains of L. turionifera are found to contain small admixture from L. minor, providing rare evidence of transfer of genetic materials in duckweed. This was discussed with respect to the hypothesis that a cross of these two gave rise to L. japonica. Moreover, the conventional maximum-likelihood phylogenetic analysis clearly recognized all the species in the three genera with high bootstrap supports. In conclusion, this work offers a basic framework for using MLST to characterize Spirodela, Landoltia and in particular Lemna strains at the species level, and to study population genetics and evolution history of natural duckweed populations.     

The Comparative Biology of Closely Related Species Living in the Same Area: V. INTER- AND INTRASPECIFIC INTERFERENCE WITHIN CULTURES OF LEMNA spp. AND SALVINIA NATANS

Studies were made of the growth of populations of Lemna minor,L. polyrrhiza, L. gibba, and Salvinia natans under controlledlaboratory conditions. The intrinsic exponential growth-ratesof the clones were determined in un-crowded cultures, and thechanges in growth-rate of self-crowding cultures were measuredand interpreted in terms of an initial exponential growth-ratefollowed by a phase of arithmetic increase in weight and followedin turn by a phase in which the death of submerged and shadedfronds caused a decline from the arithmetic rate of growth.Mean frond weight declined in self-crowding cultures (exceptof L. gibba). Mixed cultures of two species were examined under self-crowdingconditions and changes in the proportions of the species werefollowed. Whereas the total weight of mixed cultures remainedvery constant between replicates, there was wide variation inthe proportions of components. The variation in the two componentswas most closely correlated (negatively) when the struggle forexistence was most evenly balanced. The mean frond weight ofthe losing component declined during the experiments. The order of decreasing vigour of species measured by variousparameters was as follows: Relative (intrinsic) growth-rate: L. minor > S. natans > L. gibba > L. polyrrhiza Arithmetic growth-rate when crowded: S. natans > L. polyrrhiza > L. gibba > L. minor Asymptotic yield per culture: L. polyrrhiza > L. minor > S. natans > L. gibba Success in mixed cultures: The success of a species in mixture could not be predicted fromthe parameters of growth in pure culture. Morphologic featuressuch as the gibbosity of L. gibba     

Isolation and Identification of Nicotinic Acid as a Flower-Inducing Factor in Lemna

Extracts of flowering plants of the long-day plant Lemna gibbaG3 and the short-day plants Lemna paucicostata 151 and 381 weretested on L. paucicostata 151 for flower-inducing activity.Crude extracts failed to show any activity but after severalpurification steps three fractions with flower-inducing activitywere obtained. One fraction obtained from all three plants wasshown to contain nicotinic acid by mass spectroscopic and NMRspectroscopic analyses. These results raise the possibilitythat nicotinic acid may act to influence the flowering processin Lemna. (Received August 28, 1985; Accepted October 29, 1985)     

Non‐trophic effects of oribatid mites on cord‐forming basidiomycetes in soil microcosms

1. Saprotrophic cord‐forming basidiomycetes are the primary agents of decomposition in forest ecosystems. Collembola and oribatid mites affect fungal growth and foraging, and therefore decomposition, through direct mycelial grazing. 2. Grazing on the fungal species Hypholoma fasciculare, Resinicium bicolor and Phanerochaete velutina by the collembola Folsomia candida, and the oribatid mites Steganacarus magnus, Euzetes globulus and Hermannia gibba was investigated in soil microcosms. 3. Folsomia candida grazed on all fungal species: radial extent of R. bicolor, hyphal coverage of all fungal species, and fractal dimension of R. bicolor and P. velutina were all reduced. Oribatid mites did not graze the fungi but did affect mycelial morphology. Steganacarus magnus caused a reduction in the radial extent of H. fasciculare, and the hyphal coverage and fractal dimension in both H. fasciculare and R. bicolor. Euzetes globulus and H. gibba reduced the hyphal coverage of P. velutina. 4. Oribatid mites are associated with a cornucopia of chemical secretions with possible anti‐fungal properties. Chemical analysis of H. gibba opisthonotal secretions revealed four main compounds, all of which are new to the known spectrum of opisthonotal components. The most abundant was (E)‐β‐farnesene. 5. Treatment effects were species‐specific in terms of both fungal and invertebrate species. This study provides the first evidence of non‐grazing effects of oribatid mites on fungal growth and morphology. This could potentially influence the spatial organisation of mycelium in forest soils and therefore the ability of fungi to locate, colonise and decompose dead organic matter.