Nymphaeaceae

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Edward L. Schneider - One of the best experts on this subject based on the ideXlab platform.

  • Xylem of early angiosperms: Novel microstructure in stem tracheids of Barclaya (Nymphaeaceae)
    Aquatic Botany, 2009
    Co-Authors: Edward L. Schneider, Sherwin Carlquist
    Abstract:

    Pit membranes of stem tracheids of all recognized species of Barclaya, an Indomalaysian genus of Nymphaeaceae, were studied with scanning electron microscopy (SEM). Pit membranes of the tracheids are composed of two thick layers, both constructed of fibrils much larger than those of tracheary elements of angiosperms other than Nymphaeaceae. The outer (distal) layer, which comprises the continuous primary wall around the tracheids, is spongiform, perforated by porosities of relatively uniform size, and confined to or most prominent on end walls of stem tracheids. The second layer consists of thick widely spaced fibrils that are oriented axially and are laid down proximally (facing the cell lumen) to the first (outer) layer, although continuous with it. These axial fibrils are attached at their ends to the pit cavities. This peculiar microstructure is not known outside Nymphaeaceae except in Brasenia and Cabomba (Cabombaceae, Nymphaeales), and has not been previously described for Barclaya. The longitudinally oriented threads and strands in perforation plates of secondary xylem of wood and stems of a variety of primitive woody angiosperms (e.g., Illicium) are not homologous to the pit membrane structure observed in stem tracheids of Barclaya, which, like other Nymphaeaceae, has only primary xylem and no perforation plates. The tracheid microstructure reported here is different from pit structures observed in any other group of vascular plants, living or fossil. The tracheid stem microstructures of Barclaya and other Nymphaeaceae appear to be a synapomorphy of Nymphaeaceae and Cabombaceae, and need further study with respect to ultrastructure and function.

  • Do tracheid microstructure and the presence of minute crystals link Nymphaeaceae, Cabombaceae and Hydatellaceae?
    Botanical Journal of the Linnean Society, 2009
    Co-Authors: Sherwin Carlquist, Edward L. Schneider
    Abstract:

    Original scanning electron microscopy (SEM) observations are presented for stems of Brasenia schreberi and Cabomba caroliniana of Cabombaceae and three species of Trithuria of Hydatellaceae. End walls of stem tracheids of Brasenia have the same peculiar microstructure that we have reported in Barclaya, Euryale, Nuphar, Nymphaea (including Ondinea) and Victoria of Nymphaeaceae. This feature unites Cabombaceae with Nymphaeaceae. The minute rhomboidal crystals on the surfaces of stellate parenchyma cells of Brasenia reported by Solereder (1906. Oxford: University Press), but not noticed since, are figured. They are like the minute crystals of the often-mentioned astrosclereids of Nymphaeaceae. Neither of these two features has been observed in Hydatellaceae. If the absence of these two features can be confirmed, the reason may be more related to ecology, development, habit and anatomical organization than to degree of phylogenetic relationship as shown by molecular studies. Anatomical observations on the stem anatomy of Trithuria are offered on the basis of paraffin sections prepared for a paper by Cheadle & Kosakai (1975. American Journal of Botany62: 1017–1026); that study is notable for a discrepancy between an illustration of a specialized vessel element on the one hand and tabular data indicating long scalariform perforation plates on the other. Long scalariform perforation plates are mostly found in scalariformly pitted vessels of monocots, whereas the tracheary elements of Trithuria mostly have helical or annular thickenings. We were unable to demonstrate the presence of vessels in Hydatellaceae. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159, 572–582.

  • Distinctive tracheid microstructure in stems of Victoria and Euryale (Nymphaeaceae).
    Botanical Journal of the Linnean Society, 2009
    Co-Authors: Sherwin Carlquist, Edward L. Schneider
    Abstract:

    Scanning electron microscopy (SEM) photographs of thick sections from liquid-preserved stems of Victoria cruziana and Euryale ferox show accretions of coarse fibrils on pit membranes of tracheids. The first-deposited fibrils are randomly orientated; on top of them (facing the tracheid lumina) are axially orientated coarse fibrils. The two systems are interconnected. Axially orientated fibrils were more extensively observed in Euryale than in Victoria and tips of fibrils in Euryale extend over the pit apertures onto secondary wall surfaces. Tracheid–parenchyma interfaces bear rudimentary coarse fibrils on the tracheid side. End walls of Victoria tracheids have highly porose pit membranes, thinner and less complex than those of the lateral intertracheid walls. The structures reported in Victoria and Euryale are consistent with those concurrently reported for stems of other Nymphaeaceae. Although also present in Cabombaceae, the coarse fibrils are otherwise not reported for stems of angiosperms and are not yet reported in roots of any species. Pit membrane remnants in perforation plates of various woody dicotyledons represent a nonhomologous phenomenon. The accretions of coarse fibrils in stem tracheids of Nymphaeaceae do not appear to enhance conduction, although they do contain porosities interconnecting tracheids. Removal of pit membrane remnants from perforation plates of primitive dicotyledon woods by hydrolysis does, on the contrary, suggest conduction enhancement. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159, 52–57.

  • Floral development in the Nymphaeales
    International Journal of Plant Sciences, 2003
    Co-Authors: Edward L. Schneider, Shirley C. Tucker, Paula S. Williamson
    Abstract:

    Numerous phylogenetic analyses and recent paleobotanical findings continue to support the inclusion of Nymphaeales as basal or near‐basal extant angiosperms. The distinctive differences in floral morphology and habit between non‐Nymphaealian taxa of the ANITA grade compared with water lilies, nevertheless, remain perplexing. In this investigation, scanning electron microscope observations on Brasenia (Cabombaceae), Nuphar, Nymphaea, Ondinea, Victoria, and Euryale (Nymphaeaceae) are added to the observations of earlier investigations to enhance our understanding of early floral evolution and shifts in floral ontogeny and to contribute new data for improved cladistic analyses. The enlargement of flowers and amplification of floral parts distinctive in Nymphaeaceae is based on continuing surface area enlargement of the floral apex, allowing numerous organs to be produced acropetally in distinctive parastichies, compared with the ring meristem found in some primitive eudicots (Nelumbonaceae, Papaveraceae) tha...

  • Floral Development of Nelumbo nucifera (Nelumbonaceae)
    International Journal of Plant Sciences, 2000
    Co-Authors: Virginia Hayes, Edward L. Schneider, Sherwin Carlquist
    Abstract:

    The floral development of Nelumbo nucifera was compared with that noted in previous studies of Nelumbo, Nymphaeaceae, and other basal angiosperms. Important features include developmental evidence of only two sepals, development of an androecial ring meristem, and an apocarpous gynoecium composed of ascidiate carpels that become embedded in an expanded receptacle. Secretory papillate trichomes cover the stigma and line the stylar canal. The unique apocarpous gynoecium, which lacks a conical residual floral apex, and a greatly expanded receptacle distinguish Nelumbonaceae from the Nymphaeaceae, as does the distinctive androecial ring. Nelumbo is characterized by polysymmetric floral development, with some organs originating spirally (petals) and some in simultaneous whorls (stamens and carpels). This pattern of floral development, as well as the pattern of carpel closure by secretion, is common in several paleoherbs and eudicots and indicates phylogenetic affinity between Nelumbonaceae and basal angiosperm...

Sherwin Carlquist - One of the best experts on this subject based on the ideXlab platform.

  • Xylem of early angiosperms: Novel microstructure in stem tracheids of Barclaya (Nymphaeaceae)
    Aquatic Botany, 2009
    Co-Authors: Edward L. Schneider, Sherwin Carlquist
    Abstract:

    Pit membranes of stem tracheids of all recognized species of Barclaya, an Indomalaysian genus of Nymphaeaceae, were studied with scanning electron microscopy (SEM). Pit membranes of the tracheids are composed of two thick layers, both constructed of fibrils much larger than those of tracheary elements of angiosperms other than Nymphaeaceae. The outer (distal) layer, which comprises the continuous primary wall around the tracheids, is spongiform, perforated by porosities of relatively uniform size, and confined to or most prominent on end walls of stem tracheids. The second layer consists of thick widely spaced fibrils that are oriented axially and are laid down proximally (facing the cell lumen) to the first (outer) layer, although continuous with it. These axial fibrils are attached at their ends to the pit cavities. This peculiar microstructure is not known outside Nymphaeaceae except in Brasenia and Cabomba (Cabombaceae, Nymphaeales), and has not been previously described for Barclaya. The longitudinally oriented threads and strands in perforation plates of secondary xylem of wood and stems of a variety of primitive woody angiosperms (e.g., Illicium) are not homologous to the pit membrane structure observed in stem tracheids of Barclaya, which, like other Nymphaeaceae, has only primary xylem and no perforation plates. The tracheid microstructure reported here is different from pit structures observed in any other group of vascular plants, living or fossil. The tracheid stem microstructures of Barclaya and other Nymphaeaceae appear to be a synapomorphy of Nymphaeaceae and Cabombaceae, and need further study with respect to ultrastructure and function.

  • Do tracheid microstructure and the presence of minute crystals link Nymphaeaceae, Cabombaceae and Hydatellaceae?
    Botanical Journal of the Linnean Society, 2009
    Co-Authors: Sherwin Carlquist, Edward L. Schneider
    Abstract:

    Original scanning electron microscopy (SEM) observations are presented for stems of Brasenia schreberi and Cabomba caroliniana of Cabombaceae and three species of Trithuria of Hydatellaceae. End walls of stem tracheids of Brasenia have the same peculiar microstructure that we have reported in Barclaya, Euryale, Nuphar, Nymphaea (including Ondinea) and Victoria of Nymphaeaceae. This feature unites Cabombaceae with Nymphaeaceae. The minute rhomboidal crystals on the surfaces of stellate parenchyma cells of Brasenia reported by Solereder (1906. Oxford: University Press), but not noticed since, are figured. They are like the minute crystals of the often-mentioned astrosclereids of Nymphaeaceae. Neither of these two features has been observed in Hydatellaceae. If the absence of these two features can be confirmed, the reason may be more related to ecology, development, habit and anatomical organization than to degree of phylogenetic relationship as shown by molecular studies. Anatomical observations on the stem anatomy of Trithuria are offered on the basis of paraffin sections prepared for a paper by Cheadle & Kosakai (1975. American Journal of Botany62: 1017–1026); that study is notable for a discrepancy between an illustration of a specialized vessel element on the one hand and tabular data indicating long scalariform perforation plates on the other. Long scalariform perforation plates are mostly found in scalariformly pitted vessels of monocots, whereas the tracheary elements of Trithuria mostly have helical or annular thickenings. We were unable to demonstrate the presence of vessels in Hydatellaceae. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159, 572–582.

  • Distinctive tracheid microstructure in stems of Victoria and Euryale (Nymphaeaceae).
    Botanical Journal of the Linnean Society, 2009
    Co-Authors: Sherwin Carlquist, Edward L. Schneider
    Abstract:

    Scanning electron microscopy (SEM) photographs of thick sections from liquid-preserved stems of Victoria cruziana and Euryale ferox show accretions of coarse fibrils on pit membranes of tracheids. The first-deposited fibrils are randomly orientated; on top of them (facing the tracheid lumina) are axially orientated coarse fibrils. The two systems are interconnected. Axially orientated fibrils were more extensively observed in Euryale than in Victoria and tips of fibrils in Euryale extend over the pit apertures onto secondary wall surfaces. Tracheid–parenchyma interfaces bear rudimentary coarse fibrils on the tracheid side. End walls of Victoria tracheids have highly porose pit membranes, thinner and less complex than those of the lateral intertracheid walls. The structures reported in Victoria and Euryale are consistent with those concurrently reported for stems of other Nymphaeaceae. Although also present in Cabombaceae, the coarse fibrils are otherwise not reported for stems of angiosperms and are not yet reported in roots of any species. Pit membrane remnants in perforation plates of various woody dicotyledons represent a nonhomologous phenomenon. The accretions of coarse fibrils in stem tracheids of Nymphaeaceae do not appear to enhance conduction, although they do contain porosities interconnecting tracheids. Removal of pit membrane remnants from perforation plates of primitive dicotyledon woods by hydrolysis does, on the contrary, suggest conduction enhancement. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159, 52–57.

  • Floral Development of Nelumbo nucifera (Nelumbonaceae)
    International Journal of Plant Sciences, 2000
    Co-Authors: Virginia Hayes, Edward L. Schneider, Sherwin Carlquist
    Abstract:

    The floral development of Nelumbo nucifera was compared with that noted in previous studies of Nelumbo, Nymphaeaceae, and other basal angiosperms. Important features include developmental evidence of only two sepals, development of an androecial ring meristem, and an apocarpous gynoecium composed of ascidiate carpels that become embedded in an expanded receptacle. Secretory papillate trichomes cover the stigma and line the stylar canal. The unique apocarpous gynoecium, which lacks a conical residual floral apex, and a greatly expanded receptacle distinguish Nelumbonaceae from the Nymphaeaceae, as does the distinctive androecial ring. Nelumbo is characterized by polysymmetric floral development, with some organs originating spirally (petals) and some in simultaneous whorls (stamens and carpels). This pattern of floral development, as well as the pattern of carpel closure by secretion, is common in several paleoherbs and eudicots and indicates phylogenetic affinity between Nelumbonaceae and basal angiosperm...

  • vessels in Nymphaeaceae nuphar nymphaea and ondinea
    International Journal of Plant Sciences, 1995
    Co-Authors: Edward L. Schneider, Kelley Beamer, Sherwin Carlquist, Alison Kohn
    Abstract:

    Roots of Nuphar, Nymphaea, and Ondinea were studied with SEM in order to determine presence of perforations in end walls of tracheary elements. Nuphar has occasional small perforations and Nymphaea has a scattering of small perforations, but no perforations were observed in Ondinea. In Nuphar and Nymphaea, metaxylem elements have helical-reticulate wall thickenings, but end walls of tracheary elements have at least some scalariform pitting; in Ondinea, end walls are identical to side walls. In all three genera, striations (thickenings) in primary walls of tracheary elements are common, as in Euryale and Victoria.

Jan Christian Habel - One of the best experts on this subject based on the ideXlab platform.

  • ecological separation versus geographical isolation population genetics of the water lily leaf beetle galerucella nymphaeae coleoptera chrysomelidae
    Biological Journal of The Linnean Society, 2015
    Co-Authors: Robert Lechner, Ralph Kuehn, Thomas Schmitt, Jan Christian Habel
    Abstract:

    Many sedentary species with specific habitat demands occur in isolated populations and thus show strong intraspecific genetic differentiation, shaped either by geographical isolation or by local ecological adaptation to specific environmental conditions. The water-lily leaf beetle Galerucella nymphaeae has two ecotypes: one only found feeding as a specialist on just a few host plant species of the family Polygonaceae, and the other on various representatives of the family Nymphaeaceae as a generalist. To test whether the differentiation in ecotypes is also reflected in the population genetic structure, we combined amplified fragment length polymorhism data (> 500 polymorphic fragments; four sampling sites, 27–31 individuals each) with ex-situ feeding experiments (ten individuals from each site). We found that both ecotypes are genetically well differentiated. Furthermore, the genetic divergence between the two studied generalist Nymphaeaceae ecotype populations was also high. In contrast, the two specialist Polygonaceae ecotype populations clustered closely together (thus not depending on geographical isolation), indicating an ecology-driven genetic structuring. Our ex-situ reciprocal feeding experiment revealed that only individuals of the Polygonaceae ecotype are strictly dependent on their host plant family: individuals from populations feeding Polygonaceae are less likely to feed on species from the family Nymphaeaceae than the reverse. Our data show that host preference may drive genetic differentiation even more strongly than simple geographical distance.

  • Ecological separation versus geographical isolation: population genetics of the water‐lily leaf beetle Galerucella nymphaeae (Coleoptera: Chrysomelidae)
    Biological Journal of the Linnean Society, 2015
    Co-Authors: Robert Lechner, Ralph Kuehn, Thomas Schmitt, Jan Christian Habel
    Abstract:

    Many sedentary species with specific habitat demands occur in isolated populations and thus show strong intraspecific genetic differentiation, shaped either by geographical isolation or by local ecological adaptation to specific environmental conditions. The water-lily leaf beetle Galerucella nymphaeae has two ecotypes: one only found feeding as a specialist on just a few host plant species of the family Polygonaceae, and the other on various representatives of the family Nymphaeaceae as a generalist. To test whether the differentiation in ecotypes is also reflected in the population genetic structure, we combined amplified fragment length polymorhism data (> 500 polymorphic fragments; four sampling sites, 27–31 individuals each) with ex-situ feeding experiments (ten individuals from each site). We found that both ecotypes are genetically well differentiated. Furthermore, the genetic divergence between the two studied generalist Nymphaeaceae ecotype populations was also high. In contrast, the two specialist Polygonaceae ecotype populations clustered closely together (thus not depending on geographical isolation), indicating an ecology-driven genetic structuring. Our ex-situ reciprocal feeding experiment revealed that only individuals of the Polygonaceae ecotype are strictly dependent on their host plant family: individuals from populations feeding Polygonaceae are less likely to feed on species from the family Nymphaeaceae than the reverse. Our data show that host preference may drive genetic differentiation even more strongly than simple geographical distance.

María Silvia Ferrucci - One of the best experts on this subject based on the ideXlab platform.

  • Developmental and ultrastructural characters of the pollen grains and tapetum in species of Nymphaea subgenus Hydrocallis.
    Protoplasma, 2017
    Co-Authors: Lucía Melisa Zini, Beatriz G. Galati, Gabriela Zarlavsky, María Silvia Ferrucci
    Abstract:

    Variations in pollen characters and tapetum behavior were recently acknowledged in the early-divergent family Nymphaeaceae and even within the genus Nymphaea, which probably is not monophyletic; some traits such as infratectum and tapetum type are also a matter of different interpretations. In this study, developmental characters of the pollen grains and tapetum in Nymphaea subgenus Hydrocallis are provided for the first time. Observations were made in N. amazonum, N. gardneriana, and N. prolifera using light, scanning, and transmission electron microscopy. Tapetum is of the secretory type and produces orbicules. At microspore and pollen grain stages, the distal and proximal walls differ considerably. This result supports the operculate condition of the aperture in Hydrocallis, and such aperture might be plesiomorphic for Nymphaeoideae. The infratectum is intermediate, composed of inter-columellae granular elements, robust columellae consisting of agglomerated granules, complete columellae, and fused columellae. Narrow microchannels are present and persist until the mature pollen grain stage. The membranous granular layer is often present in the pollen grains of Nymphaeaceae. In N. gardneriana, this layer is most probably a component of the intine because it is lost after acetolysis. Orbicules in the Nymphaeaceae are characterized as spherical or subspherical, with a smooth sporopolleninic wall that surrounds an electron-lucent core and with individual orbicules that usually merge to give irregular aggregations. The aperture, pollen wall ultrastructure, and the tapetum of the studied species are discussed in an evolutionary and systematic context, and these characters are also compared with those of other angiosperm lineages.

  • Ultrastructural study of the female gametophyte and the epistase in Cabombaceae and Nymphaeaceae
    Flora - Morphology Distribution Functional Ecology of Plants, 2016
    Co-Authors: Lucía Melisa Zini, Beatriz G. Galati, María Silvia Ferrucci, Gabriela Zarlavsky, Sonia Rosenfeldt
    Abstract:

    Abstract Ultrastructural studies on the female gametophyte are restricted to species at relatively derived positions in the angiosperm phylogenetic tree. Therefore, this topic remains mostly unknown for the early-divergent lineages, in which a four-celled megagametophyte is common. Here, ultrastructure of the megagametophyte and micropylar nucellar epidermis was investigated in Cabomba caroliniana A. Gray (Cabombaceae), Nymphaea gardneriana Planch. and Victoria cruziana Orb. (Nymphaeaceae). The micropylar nucellar epidermis of the studied species differentiates into an epistase. These cells have metabolically active cytoplasm and thickened inner tangential walls. Epistase ultrastructure is compatible with a transfer cell specialization. This tissue may play an adaptive role in the secretion of chemotropic substances to direct the pollen tube growth toward the female gametophyte. The cytological characteristics of the female germ unit in members of Cabombaceae and Nymphaeaceae are generally similar to other angiosperms that develop a typical seven-celled, eight-nucleate female gametophyte; however, they differ in some specific points. In V. cruziana and N. gardneriana, the micropylar end of the synergids develops a rudimentary filiform apparatus with slight inward projections. By contrast, the synergids lack a filiform apparatus in C. caroliniana. Unlike most studied angiosperms, the filiform apparatus in the clade Cabombaceae-Nymphaeaceae is underdeveloped or absent, therefore character state transformations have occurred within basal angiosperms. The potential evolutionary shifts of this reproductive feature are highlighted.

Peter A. Stone - One of the best experts on this subject based on the ideXlab platform.

  • The utility of Nymphaeaceae sclereids in paleoenvironmental research
    Review of Palaeobotany and Palynology, 2012
    Co-Authors: Robby R. Marrotte, Gail L. Chmura, Peter A. Stone
    Abstract:

    Abstract As entomophilous plants, water lilies (Nymphaea) and spatterdocks (Nuphar) have low pollen production, thus can be under represented in the sediment record. These macrophytes produce distinctly shaped sclerenchyma tissue referred to as stone-cells, trichosclereids, astrosclereids or simply sclereids. This study examines the utility of using sclereids from two common species from the Nymphaeaceae Family as an alternative to their pollen. Histological studies of fresh tissues of Nymphaea odorata and Nuphar lutea show that each has distinct sclereids, but there has been confusion in the terminology used to designate their morphology. Some reports have referred to Nymphaeaceae sclereids as trichosclereids, but our histological studies show that the cells are more appropriately classified as polyramous, astrosclereids, librosclereids or rhizosclereids. Tissues from both species were treated using HCL, KOH, acetolysis and HF. We found that only the sclereids from N. lutea survived chemical treatments in a detectable form, indicating that sclereids from N. lutea can be a useful indicator of presence while the chance of observing sclereids from N. odorata in pollen preparations is very low, severely limiting the utility of the latter as a paleoecological indicator. Another limitation to using sclereids as a proxy is that they originate from tissues that require extended acetolysis treatments for release from the matrix. Finally, we examined sclereid abundance in sediments from a slough in the Florida Everglades, USA to determine if abundance of Nymphaeaceae sclereids correlates with Nymphaeaceae pollen and we find no significant correlation. Additional analyses are required to determine if empirical relationships exist amongst plant populations, pollen, sclereids and environmental conditions. A clear report of chemical treatments used and processing times are critical to verify results of studies utilizing sclereids.

  • Review of Palaeobotany and Palynology
    2012
    Co-Authors: Robby R. Marrotte, Gail L. Chmura, Peter A. Stone
    Abstract:

    abstract Article history:Received 2 June 2011Received in revised form 6 October 2011Accepted 12 October 2011Available online 18 October 2011Keywords:SclereidNymphaeaceaeNupharNymphaeaNon-pollen microfossilIdioblast As entomophilous plants, water lilies (Nymphaea) and spatterdocks (Nuphar) have low pollen production,thus can be under represented in the sediment record. These macrophytes produce distinctly shaped scleren-chyma tissue referred toasstone-cells, trichosclereids, astrosclereids or simply sclereids. Thisstudy examinesthe utility of using sclereids from two common species from the Nymphaeaceae Family as an alternative totheir pollen. Histological studies of fresh tissues of Nymphaea odorata and Nuphar lutea show that each hasdistinct sclereids, but there has been confusion in the terminology used to designate their morphology.Some reports have referred to Nymphaeaceae sclereids as trichosclereids, but our histological studies showthat the cells are more appropriately classified as polyramous, astrosclereids, librosclereids or rhizosclereids.Tissues from both species were treated using HCL, KOH, acetolysis and HF. We found that only the sclereidsfrom N. lutea survived chemical treatments in a detectable form, indicating that sclereids from N. lutea can bea useful indicator of presence while the chance of observing sclereids from N. odorata in pollen preparationsis very low, severely limiting the utility of the latter as a paleoecological indicator. Another limitation to usingsclereids as a proxy is that they originate from tissues that require extended acetolysis treatments for releasefrom the matrix. Finally, we examined sclereid abundance in sediments from a slough in the Florida Ever-glades, USA to determine if abundance of Nymphaeaceae sclereids correlates with Nymphaeaceae pollenand we find no significant correlation. Additional analyses are required to determine if empirical relation-ships exist amongst plant populations, pollen, sclereids and environmental conditions. A clear report ofchemical treatments used and processing times are critical to verify results of studies utilizing sclereids.© 2011 Elsevier B.V. All rights reserved.