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      Quillworts from the Amazon: A multidisciplinary populational study on Isoetes serracarajensis and Isoetes cangae

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          Abstract

          Isoetes are ancient quillworts members of the only genus of the order Isoetales. The genus is slow evolving but is resilient, and widespread worldwide. Two recently described species occur in the Eastern Brazilian Amazon, Isoetes serracarajensis and Isoetes cangae. They are found in the ironstone grasslands known as Canga. While I. serracarajensis is present mostly in seasonal water bodies, I. cangae is known to occur in a single permanent lake at the South mountain range. In this work, we undertake an extensive morphological, physiological and genetic characterization of both species to establish species boundaries and better understand the morphological and genetic features of these two species. Our results indicate that the morphological differentiation of the species is subtle and requires a quantitative assessment of morphological elements of the megaspore for diagnosis. We did not detect differences in microspore output, but morphological peculiarities may establish a reproductive barrier. Additionally, genetic analysis using DNA barcodes and whole chloroplast genomes indicate that although the plants are genetically very similar both approaches provide diagnostic characters. There was no indication of population structuring I. serracarajensis. These results set the basis for a deeper understanding of the evolution of the Isoetes genus.

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          Dating of the human-ape splitting by a molecular clock of mitochondrial DNA.

          A new statistical method for estimating divergence dates of species from DNA sequence data by a molecular clock approach is developed. This method takes into account effectively the information contained in a set of DNA sequence data. The molecular clock of mitochondrial DNA (mtDNA) was calibrated by setting the date of divergence between primates and ungulates at the Cretaceous-Tertiary boundary (65 million years ago), when the extinction of dinosaurs occurred. A generalized least-squares method was applied in fitting a model to mtDNA sequence data, and the clock gave dates of 92.3 +/- 11.7, 13.3 +/- 1.5, 10.9 +/- 1.2, 3.7 +/- 0.6, and 2.7 +/- 0.6 million years ago (where the second of each pair of numbers is the standard deviation) for the separation of mouse, gibbon, orangutan, gorilla, and chimpanzee, respectively, from the line leading to humans. Although there is some uncertainty in the clock, this dating may pose a problem for the widely believed hypothesis that the pipedal creature Australopithecus afarensis, which lived some 3.7 million years ago at Laetoli in Tanzania and at Hadar in Ethiopia, was ancestral to man and evolved after the human-ape splitting. Another likelier possibility is that mtDNA was transferred through hybridization between a proto-human and a proto-chimpanzee after the former had developed bipedalism.
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            Validation of the ITS2 Region as a Novel DNA Barcode for Identifying Medicinal Plant Species

            Background The plant working group of the Consortium for the Barcode of Life recommended the two-locus combination of rbcL + matK as the plant barcode, yet the combination was shown to successfully discriminate among 907 samples from 550 species at the species level with a probability of 72%. The group admits that the two-locus barcode is far from perfect due to the low identification rate, and the search is not over. Methodology/Principal Findings Here, we compared seven candidate DNA barcodes (psbA-trnH, matK, rbcL, rpoC1, ycf5, ITS2, and ITS) from medicinal plant species. Our ranking criteria included PCR amplification efficiency, differential intra- and inter-specific divergences, and the DNA barcoding gap. Our data suggest that the second internal transcribed spacer (ITS2) of nuclear ribosomal DNA represents the most suitable region for DNA barcoding applications. Furthermore, we tested the discrimination ability of ITS2 in more than 6600 plant samples belonging to 4800 species from 753 distinct genera and found that the rate of successful identification with the ITS2 was 92.7% at the species level. Conclusions The ITS2 region can be potentially used as a standard DNA barcode to identify medicinal plants and their closely related species. We also propose that ITS2 can serve as a novel universal barcode for the identification of a broader range of plant taxa.
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              Glossary of pollen and spore terminology

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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Writing – original draftRole: Writing – review & editing
                Role: Data curationRole: Formal analysis
                Role: ConceptualizationRole: Formal analysisRole: InvestigationRole: ResourcesRole: ValidationRole: VisualizationRole: Writing – original draft
                Role: ConceptualizationRole: InvestigationRole: MethodologyRole: ResourcesRole: Writing – original draft
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: ResourcesRole: Writing – original draft
                Role: Formal analysisRole: MethodologyRole: Writing – original draft
                Role: Formal analysisRole: MethodologyRole: Writing – original draft
                Role: Data curationRole: Formal analysisRole: Writing – original draft
                Role: Formal analysisRole: InvestigationRole: Writing – original draft
                Role: Formal analysisRole: InvestigationRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: InvestigationRole: ValidationRole: Writing – original draft
                Role: Formal analysis
                Role: Formal analysisRole: MethodologyRole: Writing – original draft
                Role: Formal analysisRole: Writing – original draft
                Role: ResourcesRole: Writing – original draft
                Role: Formal analysisRole: InvestigationRole: MethodologyRole: ResourcesRole: Writing – original draft
                Role: ResourcesRole: Writing – original draft
                Role: ResourcesRole: Writing – original draft
                Role: Formal analysisRole: ResourcesRole: VisualizationRole: Writing – original draft
                Role: Formal analysisRole: Funding acquisitionRole: InvestigationRole: Project administrationRole: Writing – original draft
                Role: ConceptualizationRole: Funding acquisitionRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: VisualizationRole: Writing – original draft
                Role: ConceptualizationRole: Formal analysisRole: Funding acquisitionRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                8 August 2018
                2018
                : 13
                : 8
                : e0201417
                Affiliations
                [1 ] Environmental Genomics Group, Instituto Tecnológico Vale, Belém, PA, Brazil
                [2 ] Environmental Geology and Water Resources Group, Instituto Tecnológico Vale, Belém, PA, Brazil
                [3 ] Biodiversity and Ecosystems Services Group, Instituto Tecnológico Vale, Belém, PA, Brazil
                [4 ] Environmental Technology Group, Instituto Tecnológico Vale, Belém, PA, Brazil
                [5 ] Botany Coordination, Museu Paraense Emílio Goeldi, Belém, PA, Brazil
                [6 ] Zoobotanical Park, Vale, Parauapebas, PA, Brazil
                [7 ] Environmental Studies, Amplo Engenharia, MG, Brazil
                [8 ] Environmental Studies Office, Vale, Belo Horizonte, MG, Brazil
                [9 ] North Ferrous Environmental Office, Vale, Parauapebas, PA, Brazil
                [10 ] Director, Instituto Tecnológico Vale, Belém, PA, Brazil
                Universita degli Studi di Siena, ITALY
                Author notes

                Competing Interests: The manuscript was conducted in collaboration and with financial support from Vale (project Canga Plant Genomics), the employer of TMR, TF and AC. FMGS is an employee of Amplo Engenharia who is often contracted by Vale for environmental assessment work, but not for this work. The results of this work were not directly used for any environmental licensing purposes. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.

                Author information
                http://orcid.org/0000-0002-0111-8209
                http://orcid.org/0000-0003-0054-3438
                Article
                PONE-D-18-11214
                10.1371/journal.pone.0201417
                6082551
                30089144
                374d88f4-af95-4875-8438-aa10f0a2d3b0
                © 2018 Nunes 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.

                History
                : 13 April 2018
                : 13 July 2018
                Page count
                Figures: 14, Tables: 3, Pages: 24
                Funding
                Funded by: Vale
                Award ID: Canga Plant Genomics
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100003593, Conselho Nacional de Desenvolvimento Científico e Tecnológico;
                Award ID: 307479/2016-1
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100003593, Conselho Nacional de Desenvolvimento Científico e Tecnológico;
                Award ID: 459913/2014-0
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100002322, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior;
                Award ID: 88887.141259/2017-00
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100002322, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior;
                Award ID: 88887.130628/2016-00
                Award Recipient :
                The manuscript was conducted in collaboration and with financial support from Vale (projetc Canga Plant Genomics). Additional funds were received from Brazilian Governmental agencies CNPq and CAPES. Vale provided support in the form of salaries for authors TMR, TF and AC, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Amplo Engenharia provided support in the form of salaries for author FMGS, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.
                Categories
                Research Article
                Biology and Life Sciences
                Genetics
                Genomics
                Plant Genomics
                Plant Genomes
                Chloroplast Genome
                Biology and Life Sciences
                Biotechnology
                Plant Biotechnology
                Plant Genomics
                Plant Genomes
                Chloroplast Genome
                Biology and Life Sciences
                Plant Science
                Plant Biotechnology
                Plant Genomics
                Plant Genomes
                Chloroplast Genome
                Biology and Life Sciences
                Genetics
                Plant Genetics
                Plant Genomics
                Plant Genomes
                Chloroplast Genome
                Biology and Life Sciences
                Plant Science
                Plant Genetics
                Plant Genomics
                Plant Genomes
                Chloroplast Genome
                Biology and life sciences
                Molecular biology
                Molecular biology techniques
                DNA barcoding
                Research and analysis methods
                Molecular biology techniques
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                Biology and life sciences
                Evolutionary biology
                Evolutionary systematics
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                Taxonomy
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                Molecular systematics
                DNA barcoding
                Computer and information sciences
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                Taxonomy
                Evolutionary systematics
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                Research and Analysis Methods
                Imaging Techniques
                Morphometry
                Biology and Life Sciences
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                Earth Sciences
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                Plant Science
                Plant Anatomy
                Leaves
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                Cell Biology
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                Plant Cells
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                Biology and Life Sciences
                Plant Science
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                Plant Cells
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                Biology and Life Sciences
                Genetics
                Molecular Genetics
                Biology and Life Sciences
                Molecular Biology
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                Earth Sciences
                Marine and Aquatic Sciences
                Bodies of Water
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                Earth Sciences
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                Custom metadata
                GenBank accession numbers for DNA barcodes are: atpF-atpH (MF805103 - MF805125), ITS2 (MF805393 - MF805555), trnH-psbA (MF805006 - MF805102) and psbK-psbI (MF804937 - MF804950). BOLD accession numbers are: ISO001-17 – ISO047-17, ISO049-17 – ISO064-17, ISO067-17, ISO070-17 – ISO072-17, ISO074-17 – ISO079-17, ISO081-17, ISO084-17 – ISO174-17. Chloroplast genome sequences were deposited in GenBank under accession numbers MG019393 for ITV3828 (Isoetes cangae), MG019394 for ITV2008 (Isoetes cangae), and MG019395 for ITV411 (Isoetes serracarajensis). Raw data were deposited at NCBI’s Sequence Read Archive (SRA) with the following IDs ITV2008 (SRR6941192), ITV411 (SRR6941193), and ITV3828 (SRR6941194).

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