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Klaus J. Appenroth - One of the best experts on this subject based on the ideXlab platform.
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genome and time of day transcriptome of Wolffia australiana link morphological minimization with gene loss and less growth control
Genome Research, 2020Co-Authors: Todd P Michael, Sowjanya K Sree, Evan Ernst, Nolan T Hartwick, Philomena Chu, Douglas W Bryant, Sarah Gilbert, Stefan Ortleb, Erin Baggs, Klaus J. AppenrothAbstract:Rootless plants in the genus Wolffia are some of the fastest growing known plants on Earth. Wolffia have a reduced body plan, primarily multiplying through a budding type of asexual reproduction. Here, we generated draft reference genomes for Wolffia australiana (Benth.) Hartog & Plas, which has the smallest genome size in the genus at 357 Mb and has a reduced set of predicted protein-coding genes at about 15,000. Comparison between multiple high-quality draft genome sequences from W. australiana clones confirmed loss of several hundred genes that are highly conserved among flowering plants, including genes involved in root developmental and light signaling pathways. Wolffia has also lost most of the conserved nucleotide-binding leucine-rich repeat (NLR) genes that are known to be involved in innate immunity, as well as those involved in terpene biosynthesis, while having a significant overrepresentation of genes in the sphingolipid pathways that may signify an alternative defense system. Diurnal expression analysis revealed that only 13% of Wolffia genes are expressed in a time-of-day (TOD) fashion, which is less than the typical ∼40% found in several model plants under the same condition. In contrast to the model plants Arabidopsis and rice, many of the pathways associated with multicellular and developmental processes are not under TOD control in W. australiana, where genes that cycle the conditions tested predominantly have carbon processing and chloroplast-related functions. The Wolffia genome and TOD expression data set thus provide insight into the interplay between a streamlined plant body plan and optimized growth.
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genome and time of day transcriptome of Wolffia australiana link morphological extreme minimization with un gated plant growth
bioRxiv, 2020Co-Authors: Todd P Michael, Sowjanya K Sree, Evan Ernst, Nolan T Hartwick, Philomena Chu, Douglas W Bryant, Sarah Gilbert, Stefan Ortleb, Erin Baggs, Klaus J. AppenrothAbstract:Abstract Wolffia is the fastest growing plant genus on Earth with a recorded doubling time of less than a day. Wolffia has a dramatically reduced body plan, primarily growing through a continuous, budding-type asexual reproduction with no obvious phase transition. Most plants are bound by the 24-hour light-dark cycle with the majority of processes such as gene expression partitioned or phased to a specific time-of-day (TOD). However, the role that TOD information and the circadian clock plays in facilitating the growth of a fast-growing plant is unknown. Here we generated draft reference genomes for Wolffia australiana (Benth.) Hartog & Plas to monitor gene expression over a two-day time course under light-dark cycles. Wolffia australiana has the smallest genome size in the genus at 357 Mb and has a dramatically reduced gene set at 15,312 with a specific loss of root (WOX5), vascular (CASP), circadian (TOC1), and light-signaling (NPH3) genes. Remarkably, it has also lost all but one of the NLR genes that are known to be involved in innate immunity. In addition, only 13% of its genes cycle, which is far less than in other plants, with an overrepresentation of genes associated with carbon processing and chloroplast-related functions. Despite having a focused set of cycling genes, TOD cis-elements are conserved in W. australiana, consistent with the overall conservation of transcriptional networks. In contrast to the model plants Arabidopsis thaliana and Oryza sativa, the reduction in cycling genes correlates with fewer pathways under TOD control in Wolffia, which could reflect a release of functional gating. Since TOD networks and the circadian clock work to gate activities to specific times of day, this minimization of regulation may enable Wolffia to grow continuously with optimal economy. Wolffia is an ideal model to study the transcriptional control of growth and the findings presented here could serve as a template for plant improvement.
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Duckweed for Human Nutrition: No Cytotoxic and No Anti-Proliferative Effects on Human Cell Lines
Plant Foods for Human Nutrition, 2019Co-Authors: K. Sowjanya Sree, Hans-martin Dahse, Jima N. Chandran, Bernd Schneider, Gerhard Jahreis, Klaus J. AppenrothAbstract:Duckweeds (Lemnaceae) possess good qualitative and quantitative profiles of nutritional components for its use as human food. However, no studies have been conducted on the probable presence or absence of any adverse effects. The extracts from seven duckweed species ( Spirodela polyrhiza, Landoltia punctata, Lemna gibba, Lemna minor, Wolffiella hyalina, Wolffia globosa , and Wolffia microscopica ) covering all five genera of the plant family were herewith tested for cytotoxic effects on the human cell lines HUVEC, K-562, and HeLa and for anti-proliferative activity on HUVEC and K-562 cell lines. From these assays, it is evident that duckweeds do not possess any detectable anti-proliferative or cytotoxic effects, thus, the high nutritional value is not diminished by such detrimental factors. The present result is a first step to exclude any harmful effects of highly nutritious duckweed for human.
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nutritional value of the duckweed species of the genus Wolffia lemnaceae as human food
Frontiers in Chemistry, 2018Co-Authors: Klaus J. Appenroth, Sowjanya K Sree, Manuela Bog, Josef Ecker, Claudine Seeliger, Volker Bohm, Stefan Lorkowski, Katrin Sommer, Walter Vetter, Karla TolzinbanaschAbstract:Species of the genus Wolffia are traditionally used as human food in some of the Asian countries. Therefore, all 11 species of this genus, identified by molecular barcoding, were investigated for ingredients relevant to human nutrition. The total protein content varied between 20 and 30% of the freeze-dry weight, the starch content between 10 and 20%, the fat content between 1 and 5%, and the fiber content was ~25%. The essential amino acid content was higher or close to the requirements of preschool-aged children according to standards of the World Health Organization. The fat content was low, but the fraction of polyunsaturated fatty acids was above 60% of total fat and the content of n-3 polyunsaturated fatty acids was higher than that of n-6 polyunsaturated fatty acids in most species. The content of macro- and microelements (minerals) not only depended on the cultivation conditions but also on the genetic background of the species. This holds true also for the content of tocopherols, several carotenoids and phytosterols in different species and even intraspecific, clonal differences were detected in Wolffia globosa and Wolffia arrhiza. Thus, the selection of suitable clones for further applications is important. Due to the very fast growth and the highest yield in most of the nutrients, Wolffia microscopica has a high potential for practical applications in human nutrition.
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Table_1_Nutritional Value of the Duckweed Species of the Genus Wolffia (Lemnaceae) as Human Food.DOCX
2018Co-Authors: Klaus J. Appenroth, Sowjanya K Sree, Manuela Bog, Josef Ecker, Claudine Seeliger, Volker Bohm, Stefan Lorkowski, Katrin Sommer, Walter Vetter, Karla Tolzin-banaschAbstract:Species of the genus Wolffia are traditionally used as human food in some of the Asian countries. Therefore, all 11 species of this genus, identified by molecular barcoding, were investigated for ingredients relevant to human nutrition. The total protein content varied between 20 and 30% of the freeze-dry weight, the starch content between 10 and 20%, the fat content between 1 and 5%, and the fiber content was ~25%. The essential amino acid content was higher or close to the requirements of preschool-aged children according to standards of the World Health Organization. The fat content was low, but the fraction of polyunsaturated fatty acids was above 60% of total fat and the content of n-3 polyunsaturated fatty acids was higher than that of n-6 polyunsaturated fatty acids in most species. The content of macro- and microelements (minerals) not only depended on the cultivation conditions but also on the genetic background of the species. This holds true also for the content of tocopherols, several carotenoids and phytosterols in different species and even intraspecific, clonal differences were detected in Wolffia globosa and Wolffia arrhiza. Thus, the selection of suitable clones for further applications is important. Due to the very fast growth and the highest yield in most of the nutrients, Wolffia microscopica has a high potential for practical applications in human nutrition.
Sergey Dolgov - One of the best experts on this subject based on the ideXlab platform.
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Wolffia arrhiza as a promising producer of recombinant hirudin
3 Biotech, 2021Co-Authors: Pavel Khvatkov, Alexsey Firsov, Anastasiya Shvedova, Lyubov Shaloiko, Oleg Kozlov, Mariya Chernobrovkina, Alexander Pushin, Sergey DolgovAbstract:The production of recombinant proteins in transgenic plants is becoming an increasingly serious alternative to classical biopharming methods as knowledge about this process grows. Wolffia arrhiza , an aquatic plant unique in its anatomy, is a promising expression system that can grow in submerged culture in bioreactors. In our study 8550 explants were subjected to Agrobacterium -mediated transformation, and 41 independent hygromycin-resistant Wolffia lines were obtained, with the transformation efficiency of 0.48%. 40 of them contained the hirudin-1 gene (codon-optimized for expression in plants) and were independent lines of nuclear-transformed Wolffia , the transgenic insertion has been confirmed by PCR and Southern blot analysis. We have analyzed the accumulation of the target protein and its expression has been proven in three transgenic lines. The maximum accumulation of recombinant hirudin was 0.02% of the total soluble protein, which corresponds to 775.5 ± 111.9 ng g^−1 of fresh weight of the plant. The results will be used in research on the development of an expression system based on Wolffia plants for the production of hirudin and other recombinant pharmaceutical proteins.
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Development of Wolffia arrhiza as a Producer for Recombinant Human Granulocyte Colony-Stimulating Factor
Frontiers in chemistry, 2018Co-Authors: Pavel Khvatkov, Alexsey Firsov, Anastasiya Shvedova, Lyubov Shaloiko, Oleg Kozlov, Mariya Chernobrovkina, Alexander Pushin, I. I. Tarasenko, Inna Chaban, Sergey DolgovAbstract:To date, the expression of recombinant proteins in transgenic plants is becoming a powerful alternative to classical expression methods. Special efforts are directed to the development of contained cultivation systems based on cell culture or rhyzosecretion, which reliably prevents the heterologous DNA releasing into the environment. A promising object for the development of such systems is the tiny aquatic plant of Wolffia (Wolffia arrhiza (L.) Horkel ex Wimm.), which can be used as a dipped culture in bioreactors. Herein we have expressed the human granulocyte colony-stimulating factor (hG-CSF) in nuclear-transformed Wolffia. The nucleotide sequence of hG-CSF was optimized for expression in Wolffia and cloned into the vector pCamGCSF downstream of double CaMV 35S promoter. Wolffia plants were successfully transformed and 34 independent transgenic lines with hG-CSF gene were obtained, PCR and Southern blot analysis confirmed the transgenic origin of these lines. Western blot analysis revealed accumulation of the target protein in 33 transgenic lines. Quantitative ELISA of protein extracts from these lines showed hG-CSF accumulation up to 35.5 mg/kg of Wolffia raw weight (0.194% of total soluble protein). This relatively high yield holds promise for the development of Wolffia-based expression system in strictly controlled format to produce various recombinant proteins.
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Table_1_Development of Wolffia arrhiza as a Producer for Recombinant Human Granulocyte Colony-Stimulating Factor.DOCX
2018Co-Authors: Pavel Khvatkov, Alexsey Firsov, Anastasiya Shvedova, Lyubov Shaloiko, Oleg Kozlov, Mariya Chernobrovkina, Alexander Pushin, Inna Chaban, Irina Tarasenko, Sergey DolgovAbstract:To date, the expression of recombinant proteins in transgenic plants is becoming a powerful alternative to classical expression methods. Special efforts are directed to the development of contained cultivation systems based on cell culture or rhyzosecretion, which reliably prevents the heterologous DNA releasing into the environment. A promising object for the development of such systems is the tiny aquatic plant of Wolffia arrhiza, which can be used as a dipped culture in bioreactors. Herein we have expressed the human granulocyte colony-stimulating factor (hG-CSF) in nuclear-transformed Wolffia. The nucleotide sequence of hG-CSF was optimized for expression in Wolffia and cloned into the vector pCamGCSF downstream of double CaMV 35S promoter. Wolffia plants were successfully transformed and 34 independent transgenic lines with hG-CSF gene were obtained, PCR and Southern blot analysis confirmed the transgenic origin of these lines. Western blot analysis revealed accumulation of the target protein in 33 transgenic lines. Quantitative ELISA of protein extracts from these lines showed hG-CSF accumulation up to 35.5 mg/kg of Wolffia fresh weight (0.194% of total soluble protein). This relatively high yield holds promise for the development of Wolffia-based expression system in strictly controlled format to produce various recombinant proteins.
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Transformation of Wolffia arrhiza (L.) Horkel ex Wimm
Plant Cell Tissue and Organ Culture (PCTOC), 2015Co-Authors: Pavel Khvatkov, Mariya Chernobrovkina, Alexander Pushin, Anna Okuneva, Sergey DolgovAbstract:To date, Lemna and Spirodela species of the family Lemnaceae are the only species that have been used to produce recombinant proteins for pharmaceutical and veterinary purposes. Wolffia arrhiza is the most evolutionarily advanced species of the Lemnaceae. A rootless duckweed, it is the most promising target for biopharmingas a candidate for submerged cultivation in a fermenter. As a first step toward future biotechnological use of Wolffia, we established a stable transformation system for it based on Agrobacterium-mediated transformation. Following inoculation with the bacteria, Wolffia cluster explants were cultured for 2 weeks on media containing 2.0 mg l−1 2,4-dichlorophenoxyacetic acid and 2.0 mg l−1 N6-benzyladenine. Explants were then transferred to growth regulator-free media in the presence of 5.0 mg l−1 hygromycin B to select antibiotic-resistant plants. Other selective agents—kanamycin and phosphinothricin—were not suitable for Wolffia, nor was application of particle bombardment for the delivery of foreign DNA to Wolffia explants. The developed agro transformation conditions yielded stably transformed lines of Wolffia, confirmed by Southern blotting, with an efficiency of 0.2–0.4 % transgenes per 100 explants.
Leonardo Maltchik - One of the best experts on this subject based on the ideXlab platform.
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Whole angiosperms Wolffia columbiana disperse by gut passage through wildfowl in South America
Biology letters, 2018Co-Authors: Glaura G. Silva, Andy J. Green, Vinícius Weber, P. Hoffmann, Ádám Lovas-kiss, Cristina Stenert, Leonardo MaltchikAbstract:For the first time to our knowledge, we demonstrate that whole angiosperm individuals can survive gut passage through birds, and that this occurs in the field. Floating plants of the genus Wolffia are the smallest of all flowering plants. Fresh droppings of white-faced whistling duck Dendrocygna viduata ( n = 49) and coscoroba swan Coscoroba coscoroba ( n = 22) were collected from Brazilian wetlands. Intact Wolffia columbiana were recovered from 16% of D. viduata and 32% of Coscoroba samples (total = 164 plantlets). The viability of plants was tested, and asexual reproduction was confirmed. Wolffia columbiana is an expanding alien in Europe. Avian endozoochory of asexual angiosperm propagules may be an important, overlooked dispersal means for aquatic plants, and may contribute to the invasive character of alien species.
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Supporting Information from Whole angiosperms Wolffia columbiana disperse by gut passage through wildfowl in South America
2018Co-Authors: Glaura G. Silva, Andy J. Green, Vinícius Weber, P. Hoffmann, Ádám Lovas-kiss, Cristina Stenert, Leonardo MaltchikAbstract:Supplementary Appendix to: Whole angiosperms Wolffia columbiana disperse by gut passage through wildfowl in South America.
Pavel Khvatkov - One of the best experts on this subject based on the ideXlab platform.
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Wolffia arrhiza as a promising producer of recombinant hirudin
3 Biotech, 2021Co-Authors: Pavel Khvatkov, Alexsey Firsov, Anastasiya Shvedova, Lyubov Shaloiko, Oleg Kozlov, Mariya Chernobrovkina, Alexander Pushin, Sergey DolgovAbstract:The production of recombinant proteins in transgenic plants is becoming an increasingly serious alternative to classical biopharming methods as knowledge about this process grows. Wolffia arrhiza , an aquatic plant unique in its anatomy, is a promising expression system that can grow in submerged culture in bioreactors. In our study 8550 explants were subjected to Agrobacterium -mediated transformation, and 41 independent hygromycin-resistant Wolffia lines were obtained, with the transformation efficiency of 0.48%. 40 of them contained the hirudin-1 gene (codon-optimized for expression in plants) and were independent lines of nuclear-transformed Wolffia , the transgenic insertion has been confirmed by PCR and Southern blot analysis. We have analyzed the accumulation of the target protein and its expression has been proven in three transgenic lines. The maximum accumulation of recombinant hirudin was 0.02% of the total soluble protein, which corresponds to 775.5 ± 111.9 ng g^−1 of fresh weight of the plant. The results will be used in research on the development of an expression system based on Wolffia plants for the production of hirudin and other recombinant pharmaceutical proteins.
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Development of Wolffia arrhiza as a Producer for Recombinant Human Granulocyte Colony-Stimulating Factor
Frontiers in chemistry, 2018Co-Authors: Pavel Khvatkov, Alexsey Firsov, Anastasiya Shvedova, Lyubov Shaloiko, Oleg Kozlov, Mariya Chernobrovkina, Alexander Pushin, I. I. Tarasenko, Inna Chaban, Sergey DolgovAbstract:To date, the expression of recombinant proteins in transgenic plants is becoming a powerful alternative to classical expression methods. Special efforts are directed to the development of contained cultivation systems based on cell culture or rhyzosecretion, which reliably prevents the heterologous DNA releasing into the environment. A promising object for the development of such systems is the tiny aquatic plant of Wolffia (Wolffia arrhiza (L.) Horkel ex Wimm.), which can be used as a dipped culture in bioreactors. Herein we have expressed the human granulocyte colony-stimulating factor (hG-CSF) in nuclear-transformed Wolffia. The nucleotide sequence of hG-CSF was optimized for expression in Wolffia and cloned into the vector pCamGCSF downstream of double CaMV 35S promoter. Wolffia plants were successfully transformed and 34 independent transgenic lines with hG-CSF gene were obtained, PCR and Southern blot analysis confirmed the transgenic origin of these lines. Western blot analysis revealed accumulation of the target protein in 33 transgenic lines. Quantitative ELISA of protein extracts from these lines showed hG-CSF accumulation up to 35.5 mg/kg of Wolffia raw weight (0.194% of total soluble protein). This relatively high yield holds promise for the development of Wolffia-based expression system in strictly controlled format to produce various recombinant proteins.
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Development of Wolffia arrhiza as a Producer for Recombinant Human Granulocyte Colony-Stimulating Factor
Frontiers Media S.A., 2018Co-Authors: Pavel Khvatkov, Alexsey Firsov, Anastasiya Shvedova, Lyubov Shaloiko, Oleg Kozlov, Mariya Chernobrovkina, Alexander PushinAbstract:To date, the expression of recombinant proteins in transgenic plants is becoming a powerful alternative to classical expression methods. Special efforts are directed to the development of contained cultivation systems based on cell culture or rhyzosecretion, which reliably prevents the heterologous DNA releasing into the environment. A promising object for the development of such systems is the tiny aquatic plant of Wolffia arrhiza, which can be used as a dipped culture in bioreactors. Herein we have expressed the human granulocyte colony-stimulating factor (hG-CSF) in nuclear-transformed Wolffia. The nucleotide sequence of hG-CSF was optimized for expression in Wolffia and cloned into the vector pCamGCSF downstream of double CaMV 35S promoter. Wolffia plants were successfully transformed and 34 independent transgenic lines with hG-CSF gene were obtained, PCR and Southern blot analysis confirmed the transgenic origin of these lines. Western blot analysis revealed accumulation of the target protein in 33 transgenic lines. Quantitative ELISA of protein extracts from these lines showed hG-CSF accumulation up to 35.5 mg/kg of Wolffia fresh weight (0.194% of total soluble protein). This relatively high yield holds promise for the development of Wolffia-based expression system in strictly controlled format to produce various recombinant proteins
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Table_1_Development of Wolffia arrhiza as a Producer for Recombinant Human Granulocyte Colony-Stimulating Factor.DOCX
2018Co-Authors: Pavel Khvatkov, Alexsey Firsov, Anastasiya Shvedova, Lyubov Shaloiko, Oleg Kozlov, Mariya Chernobrovkina, Alexander Pushin, Inna Chaban, Irina Tarasenko, Sergey DolgovAbstract:To date, the expression of recombinant proteins in transgenic plants is becoming a powerful alternative to classical expression methods. Special efforts are directed to the development of contained cultivation systems based on cell culture or rhyzosecretion, which reliably prevents the heterologous DNA releasing into the environment. A promising object for the development of such systems is the tiny aquatic plant of Wolffia arrhiza, which can be used as a dipped culture in bioreactors. Herein we have expressed the human granulocyte colony-stimulating factor (hG-CSF) in nuclear-transformed Wolffia. The nucleotide sequence of hG-CSF was optimized for expression in Wolffia and cloned into the vector pCamGCSF downstream of double CaMV 35S promoter. Wolffia plants were successfully transformed and 34 independent transgenic lines with hG-CSF gene were obtained, PCR and Southern blot analysis confirmed the transgenic origin of these lines. Western blot analysis revealed accumulation of the target protein in 33 transgenic lines. Quantitative ELISA of protein extracts from these lines showed hG-CSF accumulation up to 35.5 mg/kg of Wolffia fresh weight (0.194% of total soluble protein). This relatively high yield holds promise for the development of Wolffia-based expression system in strictly controlled format to produce various recombinant proteins.
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Transformation of Wolffia arrhiza (L.) Horkel ex Wimm
Plant Cell Tissue and Organ Culture (PCTOC), 2015Co-Authors: Pavel Khvatkov, Mariya Chernobrovkina, Alexander Pushin, Anna Okuneva, Sergey DolgovAbstract:To date, Lemna and Spirodela species of the family Lemnaceae are the only species that have been used to produce recombinant proteins for pharmaceutical and veterinary purposes. Wolffia arrhiza is the most evolutionarily advanced species of the Lemnaceae. A rootless duckweed, it is the most promising target for biopharmingas a candidate for submerged cultivation in a fermenter. As a first step toward future biotechnological use of Wolffia, we established a stable transformation system for it based on Agrobacterium-mediated transformation. Following inoculation with the bacteria, Wolffia cluster explants were cultured for 2 weeks on media containing 2.0 mg l−1 2,4-dichlorophenoxyacetic acid and 2.0 mg l−1 N6-benzyladenine. Explants were then transferred to growth regulator-free media in the presence of 5.0 mg l−1 hygromycin B to select antibiotic-resistant plants. Other selective agents—kanamycin and phosphinothricin—were not suitable for Wolffia, nor was application of particle bombardment for the delivery of foreign DNA to Wolffia explants. The developed agro transformation conditions yielded stably transformed lines of Wolffia, confirmed by Southern blotting, with an efficiency of 0.2–0.4 % transgenes per 100 explants.
Glaura G. Silva - One of the best experts on this subject based on the ideXlab platform.
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Whole angiosperms Wolffia columbiana disperse by gut passage through wildfowl in South America
Biology letters, 2018Co-Authors: Glaura G. Silva, Andy J. Green, Vinícius Weber, P. Hoffmann, Ádám Lovas-kiss, Cristina Stenert, Leonardo MaltchikAbstract:For the first time to our knowledge, we demonstrate that whole angiosperm individuals can survive gut passage through birds, and that this occurs in the field. Floating plants of the genus Wolffia are the smallest of all flowering plants. Fresh droppings of white-faced whistling duck Dendrocygna viduata ( n = 49) and coscoroba swan Coscoroba coscoroba ( n = 22) were collected from Brazilian wetlands. Intact Wolffia columbiana were recovered from 16% of D. viduata and 32% of Coscoroba samples (total = 164 plantlets). The viability of plants was tested, and asexual reproduction was confirmed. Wolffia columbiana is an expanding alien in Europe. Avian endozoochory of asexual angiosperm propagules may be an important, overlooked dispersal means for aquatic plants, and may contribute to the invasive character of alien species.
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Supporting Information from Whole angiosperms Wolffia columbiana disperse by gut passage through wildfowl in South America
2018Co-Authors: Glaura G. Silva, Andy J. Green, Vinícius Weber, P. Hoffmann, Ádám Lovas-kiss, Cristina Stenert, Leonardo MaltchikAbstract:Supplementary Appendix to: Whole angiosperms Wolffia columbiana disperse by gut passage through wildfowl in South America.
