Abstract
Aerenchyma is a characteristic tissue of aquatic plants, characterized by the presence of air lacunae commonly septated by diaphragms. These are formed by one or more layers of stellate cells, which allow for the passage of gases. Most species of Eleocharis grow in wet or flooded soils and have aerenchyma in their aerial stems. However, extensive studies on this structure, which could contribute to ecological and phylogenetic studies of the group, are lacking. This work describes the structure of the diaphragm in the stem of Eleocharis species and investigates the evolution of this characteristic in the genus. Fifty-three species were analyzed under light and scanning electron microscopy. We analyzed the evolution of the characteristics by reconstructing their ancestral states based on the previously published original phylogeny. The diaphragms in Eleocharis vary mainly in the number of layers, cell shape, and cell wall thickness. The typical diaphragm of the genus is composed of three to four layers of stellate cells, with microprojections and secretory cells. The diaphragm of the group’s ancestor had practically the same characteristics as the genus’s typical diaphragm.
Key words:
anatomy; aquatic macrophytes; evolution; micromorphology; stellate cells
Resumo
Aerênquima é um tecido comum nas plantas aquáticas, caracterizado pela presença de lacunas de ar comumente septadas por diafragmas. Esses, são formados por uma ou mais camadas de células, geralmente braciformes, que permitem a passagem de gases. A maioria das espécies de Eleocharis cresce em solos úmidos ou inundados e apresenta aerênquima no caule. No entanto, essa estrutura carece de estudos amplos que poderiam contribuir aos estudos ecológicos e filogenéticos do grupo. Este trabalho descreve a estrutura do diafragma no caule de espécies de Eleocharis e investiga a evolução desse caractere no gênero. 53 espécies foram analisadas em microscopia de luz e eletrônica de varredura através de secções transversais e longitudinais do caule. A análise da evolução dos caracteres foi feita pela reconstrução de estado ancestral, baseada na filogenia original anteriormente publicada. Os diafragmas em Eleocharis variam principalmente em número de camadas, formato das células e espessura da parede celular. O diafragma típico do gênero é composto de três a quatro camadas de células braciformes lobadas, com microprojeções e células secretoras. O diafragma do ancestral do grupo apresentava praticamente as mesmas características do diafragma típico para o gênero.
Palavras-chave:
anatomia; macrófitas aquáticas; evolução; micromorfologia; célula braciforme
Introduction
Aerenchyma is a typical tissue in aquatic plants, characterized by large air spaces between the cells. Its main function is to mitigate the lack of oxygen in flooded regions (Justin & Armstrong 1987Justin SHFW & Armstrong W (1987) The anatomical characteristics of roots and plant response to soil flooding. New Phytologist 106: 465-495.); however, it also contributes to the circulation of CO2 during photosynthesis (Constable et al. 1992Constable JVH, Grace JB & Longstreth DJ (1992) High carbon dioxide concentrations in aerenchyma of Typha latifolia. American Journal of Botany 79: 415-418.; Li & Jones 1995Li M & Jones MB (1995) CO2 and O2 transport in the aerenchyma of Cyperus papyrus L. Aquatic Botany 52: 93-106.) and the circulation of hormones such as ethylene (He et al. 1996He CJ, Finlayson SA, Drew MC, Jordan WR & Morgan PW (1996) Ethylene biosynthesis during aerenchyma formation in roots of maize subjected to mechanical impedance and hypoxia. Plant Physiology 112: 1679-1685. ). The aerenchyma forms both due to cellular lysis and without it (Seago et al. 2005Seago JL, Marsh LC, Stevens KJ, Soukup A, Votrubova O & Enstone DE (2005) A re-examination of the root cortex in wetland flowering plants with respect to aerenchyma. Annals of Botany 96: 565-579.). Commonly, the aerenchyma that forms without cell lysis has large gaps septated by diaphragms.
The diaphragms are formed by one or more layers of cells that delimit the gaps, maintain intercellular spaces, and play an important role in maintaining structure and metabolic balance (Sculthorpe 1967Sculthorpe CD (1967) The biology of aquatic vascular plants. Edward Arnold, London. 610p. ). These structures allow for the continuous flow of gases, provide resistance and mechanical stabilization to the organ, restrict water entry when insect damage occurs, and provide a lateral transport pathway through the cortex, allowing for the storage of substances such as tannins and starches (Snow 1914Snow LM (1914) Contributions to the knowledge of the diaphragms of water plants I. Scirpus validus. Botanical Gazette 58: 495-517.; Sculthorpe 1967; Kaul 1971Kaul RB (1971) Diaphragms and aerenchyma in Scirpus validus. American Journal of Botany 58: 808- 816., 1972; Dickison 2000Dickison WC (2000) Integrative plant anatomy. Academic Press, San Diego. 355p.). Armstrong (1979Armstrong AC (1979) Aeration in higher plants. Advances in Botanical Research 7: 225-332. ) adds that wall thickness, porosity (intercellular spaces), and the distribution of diaphragms also influence the circulation of gases.
Eleocharis species have aerial upright stems that originate from a congested, rhizomatous, or stoloniferous stem (Trevisan & Boldrini 2008Trevisan R & Boldrini II (2008) O gênero Eleocharis R. Br. (Cyperaceae) no Rio Grande do Sul, Brasil. Revista Brasileira de Biociências 6: 7-67.). The upright stems are leafless and photosynthetic. Due to the small size and simplicity of the plant, only a few morphological features are available for phylogenetic analyses that may aid in infrageneric classification. The use of anatomical characteristics complements the morphological and molecular analyses, helping to clarify the relationships between the subgenera (Hinchliff & Roalson 2009Hinchliff CE & Roalson EH (2009) Stem architecture in Eleocharis Subgenus Limnochloa (Cyperaceae): evidence of dynamic morphological evolution in a group of pantropical sedges. American Journal of Botany 96: 1487-1499.).
Despite these efforts, the phylogenetic studies of Eleocharis show that the most recent classification by González-Elizondo & Peterson (1997González-Elizondo MS & Peterson PM (1997) A classification of and key to the supraspecific taxa in Eleocharis (Cyperaceae). Taxon 46: 433-449.) does not reflect the phylogenetic relationships within the group (Roalson & Friar 2000Roalson EH & Friar EA (2000) Infrageneric classification of Eleocharis (Cyperaceae) revisited: evidence from the internal transcribed spacer (ITS) region of nuclear ribosomal DNA. Systematic Botany 25: 323-336.; Yano et al. 2004Yano O, Katsuyama T, Tsubota H & Hoshino T (2004) Molecular phylogeny of Japanese Eleocharis (Cyperaceae) based on ITS sequence data, and chromosomal evolution. Journal of Plant Research 117: 409-419.; Roalson et al. 2010). According to Roalson et al. (2010), most of the seven clades that group the species of Eleocharis together are well supported, but some groups still show variations in their positions in the analyses, mainly those of cosmopolitan distributions. For this reason, the previously cited authors emphasize that a satisfactory classification that delimits these groups has not yet been found and that future research in search of new characters for analysis is therefore necessary. It is known that most species of Eleocharis preferentially grow in environments with wet or flooded soils and can be found either totally or partially submerged (Trevisan & Boldrini 2008Trevisan R & Boldrini II (2008) O gênero Eleocharis R. Br. (Cyperaceae) no Rio Grande do Sul, Brasil. Revista Brasileira de Biociências 6: 7-67.). Due to this plasticity in the environmental conditions needed for survival and reproduction, many emerging or submerged species present with highly developed aerenchyma tissue, both in the root and in the stem (Marcondes et al. 2021Marcondes JPBACM, Trevisan R, Chiara-Moço MC & Bona C (2021) Diversity and evolution of stem structure in Eleocharis (Cyperaceae). Rodriguesia 72: 1-13. ).
The stem aerenchyma in Eleocharis is composed of air cavities, which are often septated by diaphragms (Metcalfe 1971Metcalfe CR (1971) Anatomy of the Monocotyledons: V Cyperaceae. Ed. Oxford, Oxford. 596p.; Marcondes et al. 2021Marcondes JPBACM, Trevisan R, Chiara-Moço MC & Bona C (2021) Diversity and evolution of stem structure in Eleocharis (Cyperaceae). Rodriguesia 72: 1-13. ). According to the distribution of the cavities, two types of stem architecture are found in the subgenus Limnochloa species: spongy and septated (Hinchliff & Roalson 2009Hinchliff CE & Roalson EH (2009) Stem architecture in Eleocharis Subgenus Limnochloa (Cyperaceae): evidence of dynamic morphological evolution in a group of pantropical sedges. American Journal of Botany 96: 1487-1499.). The spongy stem presents with several air columns in the central region of the organ, intersected transversally by diaphragms, while the septated stem has a central air column, which occupies most of the organ and is interrupted by large, transverse diaphragms (Marcondes et al. 2021). These two patterns were recorded in 68 Eleocharis species, wherein spongy was the more frequent and was subdivided into two types by Marcondes et al. (2021).
Studies on the shape and anatomy of the stem have not detailed the structures of the diaphragms, which represent a potential source of characteristics that could help delimit the clades, subclades, and even problematic species of the genus. Since the stem in Eleocharis is the main photosynthetic organ and consists predominantly of aerenchyma with diaphragms, we believe that this tissue can provide morphological variations that could help in the characterization of the groups, as well as help us to understand the phylogenetic relationships of the genus. Thus, in the present, we aim to describe the anatomy and micromorphology of the diaphragms present in the aerial stem aerenchyma in representatives of four of the seven phylogenetic clades indicated for this genus (Roalson et al. 2010Roalson EH, Hinchliff CE, Trevisan R & Silva CRM (2010) Phylogenetic relationships in Eleocharis (Cyperaceae): C4 photosynthesis origins and patterns of diversification in the spikerushes. Systematic Botany 35: 257-271.) to verify the existence of anatomical characteristics exclusive to each clade, and to describe the evolutionary history of the characteristics of the genus.
Material and Methods
We analyzed 53 species of Eleocharis (Tab. 1). When available, each species was represented by three samples from different locations and environments. The samples were obtained from fixed and herborized material. Species from four clades (Roalson et al. 2010Roalson EH, Hinchliff CE, Trevisan R & Silva CRM (2010) Phylogenetic relationships in Eleocharis (Cyperaceae): C4 photosynthesis origins and patterns of diversification in the spikerushes. Systematic Botany 35: 257-271.) representing all types of aerenchyma described in Marcondes et al. (2021Marcondes JPBACM, Trevisan R, Chiara-Moço MC & Bona C (2021) Diversity and evolution of stem structure in Eleocharis (Cyperaceae). Rodriguesia 72: 1-13. ) were included. The exsiccates of the analyzed species were deposited into the FLOR, MBM, and UPCB herbaria (acronyms from Thiers, continuously updated). The seven morpho-anatomical characteristics and their variations (Tab. 2) were collected by light microscopy and scanning electron microscopy. We measured the height of the species (aerial part including stem) in about five individuals per species collected or from herbarium samples and literature data for the species not collected. The species analyzed were divided into small (< 17 cm), medium (> 17 ≤ 40 cm), and high (> 40 cm) groups (Marcondes et al. 2021) (Tab. 2).
Eleocharis (Cyperaceae) species used in the characterization of the diaphragms present in the stem aerenchyma.
Relationship of the characters of the stem diaphragm of the Eleocharis species (Cyperaceae), and their respective states. 1. Presence of secretory cells: 0 = absent; 1 = present; 2. Morphology of the diaphragm cells: 0 = lobed stellate cells; 1 = simple stellate cell; 2 = non-stellate cell; 3. Presence of microprojections on the wall of the diaphragm cells: 0 = absent; 1 = present; 4. Number of predominant layers in the diaphragm: 0 = (≤ 2); 1 = (3 to 4); 2 = (> 4); 5. Thickness of the walls of the diaphragm cells: 0 = thin; 1 = thick; 6. Morphology of the diaphragm cells of the lateral gaps: 0 = lobed stellate cells; 1 = simple stellate cells; 2 = non-stellate cells; 7. Species height: 0 = (low ≤ 16); 1 = (mean > 17 ≤ 40); 2 = (high > 40).
Microscopic analysis
Anatomical analyses of the aerenchyma were performed with material fixed in FAA70 (Johansen 1940Johansen DA (1940) Plant microtechnique. McGraw-Hill, New York. 523p.) and exsiccated material rehydrated with 30% ammonium hydroxide for three hours (Toscano de Brito 1996Toscano de Brito ALV (1996) The use of concentrated ammonia as an excellent medium for the restoration of orchid pollinaria - an example from the Ornithocephalinae. Lindleyana 11: 205-210.). After fixation and hydration, both materials were stored in 70% ethanol. For all species, transverse and longitudinal sections were made in the mid and basal regions of the organ. Permanent slides were prepared from material processed and embedded in Historesin Leica®, following the manufacturer’s guidelines. The blocks were sectioned using a 7-μm-thick rotating microtome, and the blades were stained with Toluidine Blue (O’Brien et al. 1964O’Brien TP, Feder N & Mccully ME (1964) Polychromatic staining of plant cell walls by Toluidine Blue O. Protoplasma 59: 368-373.). Ferric chloride 10% (Johansen 1940) was used to identify phenolic compounds, and we used Lugol (Johansen 1940) to detect starch grains. The analyses were performed under an Olympus microscope (BX41TF) with an attached camera (Olympus SC30) for image capture. Measurements were made under a microscope with an ocular fitted with a calibrated scale via a Zeiss 5 + 100/100 micrometric blade.
For scanning electron microscopy (SEM) analysis, the fixed or hydrated samples, as described above, were dehydrated in an ethylic series and subjected to the CO2 critical point method (BAL-TEC CPD030 Critical Point Dryer), adhered in metallic support with adhesive copper tape and metalized with gold (BALZERS SCD030). The analyses were done using the scanning electron microscope (JEOL JSM-6360LV Scanning Electron Microscope) at the Centro de Microscopia Eletrônica of UFPR (CME).
Phylogenetic analysis
The reconstruction of the ancestral states by parsimony was analyzed to evaluate the evolution of the characteristics resulting from the anatomical diagnosis. We employed the strict consensus tree used by Marcondes et al. (2021Marcondes JPBACM, Trevisan R, Chiara-Moço MC & Bona C (2021) Diversity and evolution of stem structure in Eleocharis (Cyperaceae). Rodriguesia 72: 1-13. ) in the analysis of Eleocharis stem aerenchyma patterns; this tree was generated in the TNT (Tree analysis using New Technology) with ITS1, 5.8S, and ITS2 nuclear DNA sequences and trnC-ycf6 and ycf6-psbM from the chloroplast DNA. The reconstruction was done by parsimony in the program Mesquite v. 3.03 (Maddison & Maddison 2015Maddison WP & Maddison DR (2015) Mesquite: a modular system for evolutionary analysis. Version 3.03. Available at <Available at https://www.mesquiteproject.org/ >. Access in September 2016.
https://www.mesquiteproject.org/...
). The characteristics were coded discretely, and their character states are presented in Table 2.
Results
In Eleocharis stems, gaps of different dimensions are transversely divided by diaphragms with one or more layers of cells (Fig. 1a-d). In spongy-pattern aerenchyma, these layers are arranged at different heights in the stem (Fig. 1a-b); in septate-pattern aerenchyma, the layers divide the stem into segments with wide central diaphragms (Fig. 1c). The cells that make up the diaphragms are generally stellate (Fig. 1c,e-f) with different morphologies according to the species. The diaphragms differ in the number of cell layers, size and shape of the intercellular spaces, cell morphology, wall thickness, microprojection presence, and presence of secretory cells with phenolic compounds (Tab. 2). The presence of starch (Fig. 1g) was the only characteristic that diverged between the mid and basal regions of the stem as it was present in the base but absent in the mid-region.
The number of cell layers in the diaphragm varies between species and can be thin, with up to two layers (Fig. 1h); medium, with three to four layers (Fig. 1g, i); or thick, with more than four layers (Fig. 1j-k). Although the thickness of diaphragm layers was slightly variable in most species, both thick and thin diaphragms occurred in some species (Elecharis bonariensis, E. densicaespitosa, and E. riograndensis) (Fig. 2). The thickest diaphragm was recorded in E. endounifascis, with approximately 30 layers. Clades 1 and 7 present species with all three states of the character clade 6 contains species with medium and thin diaphragms; and clade 4 contains only species with thin diaphragms (Fig. 2).
The cells of the diaphragm layers are superimposed, which allows for continuous intercellular spaces between the layers (Figs. 1k; 3a). The intercellular spaces may be conspicuous or not (Figs. 1e-f; 3a-f). The diaphragm cells of the analyzed species may be stellate (Figs. 1e-f; 3a) or nonstellate (Fig. 3f). Stellate cells are the most frequent morphology, and in general, these have six or eight extensions. The intercellular spaces are usually delimited by the extensions of three neighboring cells, forming a space that is approximately triangular (Figs. 1e; 3a) or rounded (Figs. 1f; 3b).
Stellate cells can be divided into two types according to the format of the extensions:
I - Lobed stellate cell. In this morphology, the end of the cell extension is dilated at the attachment site with the neighboring cell (Fig. 3a-f). The dilatation is variable in each species and may be discrete (Fig. 3a) or more pronounced (Fig. 3b). In diaphragms whose cell extensions are very dilated at their extremities, the intercellular spaces are reduced (Fig. 3c-d). This dilation can further branch out and give rise to cells with complex shapes (Fig. 3d-e). In most species, these cells have microprojections on the extension walls, especially in the region of the connections among cells (Fig. 3b-d). These microprojections may or may not be linked to the microprojections of neighboring cells (Fig. 3c). Stellate cells with short and dilated extensions were recorded only in the diaphragms of E. elegans, E. montana (Fig. 3f), and E. sellowiana. In the other species, the extensions were well-developed, as in E. acicularis (Fig. 3a), E. acutangula, and E. subarticulata, among others. The other clades have both states among their species (Fig. 4), except clade 4, in which all analyzed species have microprojections in the cells.
a-k. Diversity in the diaphragm cells in Eleocharis stems; MEV (a, b, g-k), light microscopy (c-f); cross sections (a, b, c, e, f) and longitudinal sections (d, g-k) - a. general view of the spongy pattern aerenchyma, E. acutangula; b. gaps with diaphragm, E. subarticulata; c. stem with small peripheral gaps (arrow) and a large central gap with diaphragms (septate pattern), phenolic idioblasts, E. endounifaceis; d. a gap cross divided by the diaphragm, E. acutangula; e-f. diaphragms in front view with stellate cells - e. E. interstincta; f. E. kuruguwai; g. diaphragm medium with three layers of cells, the central one with thick walls and the periphery with thin walls, E. mamillata; h. thin diaphragm, with a layer of thin-walled cells, E. riograndensis; i. diaphragm medium with four layers, the two thick central, E. palustris; j-k. thick diaphragms, with a number of layers greater than five; j. little thick cells walls, E. rostelata; k. very thick cell walls, E. equisetoides. (Arrow = thin-walled cell; tw = cell of thick walls; s = starch; * = phenolic idioblasts; di = diaphragm). Scale bars: a = 500 µm; b, d = 100 µm; c = 200 µm, e, f = 50 µm, g = 5 µm, h, i, j = 10 µm, k = 20 µm.
Reconstruction of the ancestral state in Eleocharis, by the method of parsimony (Mesquite v. 3.03), based on the strict consensus tree by the TNT program (v.1.1); number of layers of the stem diaphragm, black (less than or equal to two), white (three to four), striped (greater than four) and X (not analyzed). The indicated clades were previously defined based on the phylogeny of the group (Roalson et al. 2010Roalson EH, Hinchliff CE, Trevisan R & Silva CRM (2010) Phylogenetic relationships in Eleocharis (Cyperaceae): C4 photosynthesis origins and patterns of diversification in the spikerushes. Systematic Botany 35: 257-271.).
II - Simple stellate cell. In this morphology, the extensions of the cells do not have dilations, and in general, the microprojections are absent (Fig. 5a-b). These cells were recorded in the diaphragms of E. cylindrica, E. elegans, E. erythropoda, E. filiculmis, E. geniculate, E. kuroguwai, E. maculosa, E. montana (central lacuna), and E. sellowiana. In E. kuroguwai, the extensions of the stellate cells have little delimitation, creating circular intercellular spaces (Fig. 5b).
Nonstellate cells have irregular shapes on the transverse axis, are flattened on the longitudinal axis, and lack extensions and microprojections (Fig. 5c-d). These cells are arranged in a juxtaposed way, which results in nonobvious intercellular spaces. Nonstellate cells were found only in the diaphragms of small species (E. exigua, E. dunensis, E. niederleinii, E. rabenii, and E. riograndensis), and phenolic compounds may be present (Fig. 5c). Clade 7 was the only one containing all three states of the described diaphragm cell morphology, while the three other analyzed clades had species with lobed stellate cells and simple stellate cells (Fig. 6).
Diaphragm cells may have thin primary walls and lignified secondary walls of different thicknesses (Fig. 1e-k). In diaphragms with thick-walled cells, these are in the center of the layer, coated by thin-walled cells that are often torn or stretched (Figs. 1i, k; 3a-b). In diaphragms with lobed thick-walled cells, thin-walled cells may or may not exhibit microprojections. With the exception of E. kleinii, in which it was not possible to analyze this characteristic, the other species of clade 1 presented with thick-walled cells. The other clades presented predominantly thick-walled cells and isolated species or small groups with cells without any thickening in their diaphragms (Fig. 7). A thickened wall is the plesiomorphic state of the group, and the absence of thickening is an apomorphic state that appeared at least eight times in Eleocharis’s history (Fig. 7).
Most of the analyzed species presented phenolic content secretory cells (as shown by a positive test for ferric chloride) distributed randomly in the diaphragm (Tab. 2; Fig. 5c). The presence of secretory cells is the plesiomorphic state of the genus, while the absence of these cells is an apomorphic state that arose at least five times. Clades 1, 6, and 7 each contained species with and without secretory cells, while species in clade 4 all have secretory cells. The nine analyzed species presented specimens with both states (Tab. 2).
Discussion
The diaphragms in Eleocharis are composed of one to several layers of parenchyma cells, in line with the findings of Metcalfe (1971Metcalfe CR (1971) Anatomy of the Monocotyledons: V Cyperaceae. Ed. Oxford, Oxford. 596p.). The ancestral state of the number of layers of the diaphragm was ambiguous; the oldest ancestor of the group could have had either medium or thick diaphragms. Fine diaphragms compose an apomorphic state in the group, which has appeared several times in the history of the genus (see Fig. 2).
The vast majority of species present lobed stellate cells. The first records of lobed stellate cells in the genus occurred in the species E. acutangula, E. geniculata, E. intermedia, E. kleinii, E. montana, E. mutata, E. palustris, and E. subarticulata (Metcalfe 1971Metcalfe CR (1971) Anatomy of the Monocotyledons: V Cyperaceae. Ed. Oxford, Oxford. 596p.). The morphological character of the diaphragm cells demonstrates how the ancestral state influences the lobed stellate cell. Simple stellate cells and nonstellate cells are apomorphic states, where the first appeared at least nine times, and the second appeared two times throughout the evolutionary history of the group (see Fig. 6).
Most species only have one type of morphology in their diaphragm cells, but some have different morphological types in the same individual or among specimens. This is the case for E. montana and E. elegans, which present stems with septated architectures, and the morphology of the lateral diaphragm cells (present in the peripheral gaps) differs from that of the central gap diaphragm (Marcondes et al. 2021Marcondes JPBACM, Trevisan R, Chiara-Moço MC & Bona C (2021) Diversity and evolution of stem structure in Eleocharis (Cyperaceae). Rodriguesia 72: 1-13. ).
a-f. Morphological variation of the stellate cells, in MEV (a-d, f) and light microscopy (e) - a. stellate cells with free intercellular space, E. acicularis; b. stellate cells delimiting intercellular space partially obstructed by the dilatations of the lobes, E. subarticulata; c. detail of connections in dilated regions E. subarticulata; d-e. cell with branched dilatations, E. niederleinii; f. detail of the short and dilated arms of the cells with arms. (sc = stellate cells; tw = thin-walled cells; di = expansion of the dilations; mi = microprojection). Scale bars: a, b, f = 10 µm; c = 2 µm; d = 5 µm; e = 20 µm.
The intercellular spaces of the species’ diaphragms are triangular, as reported by Govindarajalu (1975Govindarajalu E (1975) The systematic anatomy of South Indians Cyperaceae: Eleocharis R. Br., Rhynchospora Vahl and Scleria Bergius. Adansonia 14: 581-632.), circular, or may not be visible. The variation of the spaces is a result of the combination of morphology, wall thickness, and cellular arrangement. Diaphragms with larger intercellular spaces are commoner in large species than in small ones. The explanation for this characteristic is given by Snow (1914Snow LM (1914) Contributions to the knowledge of the diaphragms of water plants I. Scirpus validus. Botanical Gazette 58: 495-517.), which corroborates the results of a biomechanical study by Schwendener (Schwendener apud Snow 1914), which relates the size of the extensions to the disparity in the speed of growth between the cells. While the stellate cells assume this morphology due to the tension they undergo during their growth, the surrounding tissue develops faster. As the growth of the diaphragm cells cannot keep pace with the external cells, they are stretched, forming the “arms” or extensions.
Reconstruction of the ancestral state in Eleocharis, by the method of parsimony (Mesquite v. 3.03), based on the strict consensus tree by the TNT program (v. 1.1); presence of microprojections in the cell of stem diaphragm; black (present), white (absent), and X (not analyzed). The indicated clades were previously defined based on the phylogeny of the group (Roalson et al. 2010Roalson EH, Hinchliff CE, Trevisan R & Silva CRM (2010) Phylogenetic relationships in Eleocharis (Cyperaceae): C4 photosynthesis origins and patterns of diversification in the spikerushes. Systematic Botany 35: 257-271.).
Diaphragms with thick-walled cells are present in most species, but diaphragms with thin-walled cells usually occur in smaller species. The medium to large species present a greater anatomical variety in the diaphragm and have a greater number of layers and greater wall thickness. This result is expected since one of the functions of the diaphragm is to provide mechanical resistance and stabilization to the organ (Snow 1914Snow LM (1914) Contributions to the knowledge of the diaphragms of water plants I. Scirpus validus. Botanical Gazette 58: 495-517.; Sculthorpe 1967Sculthorpe CD (1967) The biology of aquatic vascular plants. Edward Arnold, London. 610p. ; Dickison 2000Dickison WC (2000) Integrative plant anatomy. Academic Press, San Diego. 355p.; Kaul 1971Kaul RB (1971) Diaphragms and aerenchyma in Scirpus validus. American Journal of Botany 58: 808- 816., 1972). Thus, the larger stems have more developed diaphragms and thicker walls.
The morphological variations found in the shape of the diaphragm cells in Eleocharis were similarly described in other species (Snow 1914Snow LM (1914) Contributions to the knowledge of the diaphragms of water plants I. Scirpus validus. Botanical Gazette 58: 495-517.; Govindarajalu 1975Govindarajalu E (1975) The systematic anatomy of South Indians Cyperaceae: Eleocharis R. Br., Rhynchospora Vahl and Scleria Bergius. Adansonia 14: 581-632.; Bona & Alquini 1995aBona C & Alquini Y (1995a) Anatomia foliar de Hydrocleis nymphoides (Humb. & Bomp. ex Willd) Buchenau (Limnocharitaceae). Arquivos de Biologia e Tecnologia 38: 869-877., b). This result demonstrates that diaphragms, although having characteristics common to species, such as the presence of stellate cells, may have individual peculiarities or peculiarities in groups of species (i.e., the branching of the extensions). The distinctive shape found in E. kuroguwai (Fig. 5b), which gives rise to circular intercellular spaces, is an example of a morphology that was recorded in this species and E. sphacelata R. Br. (Sorrell et al. 1997Sorrell BK, Hans B & Philip TO (1997) Eleocharis sphacelata: internal gas transport pathways and modelling of aeration by pressurized flow and diffusion. New Phytol 136: 433-442.). Diaphragms without stellate cells, as reported here and by Metcalfe (1971Metcalfe CR (1971) Anatomy of the Monocotyledons: V Cyperaceae. Ed. Oxford, Oxford. 596p.), also delimit a small group of species among those analyzed (E. exigua, E. dunensis, E. niederleinii, E. rabenii, and E. riograndensis), all of which are medium-sized and small (Tab. 2). Unlike the diaphragms of some species that present vascular bundles (Kaul 1971Kaul RB (1971) Diaphragms and aerenchyma in Scirpus validus. American Journal of Botany 58: 808- 816.), the analyzed species of Eleocharis all lack these bundles.
The presence and function of microprojections in diaphragm cells have not yet been investigated in academic work. Studies of their ontogenesis are needed to understand their development and function. We can affirm that these structures are associated with lobed stellate cells in Eleocharis. They are often united with the microprojections of neighboring cells, a characteristic that is present in most species, genetically well-fixed, and can be used for taxonomic purposes. Microprojections in stellate cells have been present throughout the evolutionary history of the genus since its earliest ancestor. Our results demonstrate that the absence of microprojections appeared independently at least eight times in the group’s history (see Fig. 4).
Diaphragms may contain chloroplasts, laticifers, and starch (Snow 1914Snow LM (1914) Contributions to the knowledge of the diaphragms of water plants I. Scirpus validus. Botanical Gazette 58: 495-517.). Secretory cells in the stem are mentioned in several species of Eleocharis (Metcalfe 1971Metcalfe CR (1971) Anatomy of the Monocotyledons: V Cyperaceae. Ed. Oxford, Oxford. 596p.), may also appear in the stolon and rhizome (Hess 1953Hess H (1953) Über die Gattungen Heleocharis und Carex aus dem unteren Kongo. Berichte der Schweizerischen Botanischen Gesellschaft 63: 317-359.), and, according to Govindarajalu (1975Govindarajalu E (1975) The systematic anatomy of South Indians Cyperaceae: Eleocharis R. Br., Rhynchospora Vahl and Scleria Bergius. Adansonia 14: 581-632.), may indicate the presence of tannins. In Eleocharis, the constant presence of phenolic compounds is not of taxonomic importance but is a function of herbivory prevention (Coley 1988Coley PD (1988) Effects of plant growth rate and leaf lifetime on the amount and type of anti-herbivore defense. Oecologia 74: 531-536.) and resistance to infection by microorganisms such as fungi and bacteria (Levit 1971Levit DA (1971) Plant phenolics: an ecological perspective. The American Naturalist 105: 157-181.), which are common in these species’ aquatic environments.
a-d. Diaphragm cell morphology in Eleocharis stems; in MEV (a, b) and light microscopy (c, d) - a-b. stellate cells with simple extensions - a. straight extensions, E. montana; b. cell with body and a little distinct extensions, E. kuroguwai; c-d. non-stellate cell - c. diaphragm in frontal view showing the shape of the cells slightly irregular and juxtaposed, with phenolic idioblasts (*), E. rabenii; d. longitudinal aspect of the diaphragm, E. dunensis. (sc = stellate cell; ns = non-stellate cell; ex = extensions of the cells). Scale bars: a = 5 µm; b = 25 µm; c = 10 µm; d = 20 µm.
No unique anatomical characteristics were found among the members of the same clade. Some identified characteristics were frequently present within a genus, while others appeared more discretely. The presence of microprojections in the diaphragm cells and the thickening of their walls are the most relevant characteristics to aid in a genus’s classification, as they do not show variations within a species. Both can delimit small groups in the different clades, such as those cited in the results and exemplified in the characteristic reconstruction trees.
The evolutionary history of the diaphragm in Eleocharis reveals the genus’s anatomical variations; however, the plesiomorphic states of the analyzed characteristics are still the most common among its species. The typical Eleocharis diaphragm comprises thick-walled, lobed stellate cells with microprojections, secretory cells, and more than three layers of cells. None of the species studied presented all the characteristics’ apomorphic states. In the evolution of all the analyzed characteristics, the apomorphic state is the simplest anatomy, which demonstrates that the structure of the diaphragm is simplifying, which favors the species by reducing their energy expenditure in forming diaphragms.
Reconstruction of the ancestral state in Eleocharis, by the parsimony method (Mesquite v. 3.03), based on the strict consensus tree by the TNT program (v.1.1); Morphology of the stem diaphragm cell; black (simple stellate cell), white (lobed stellate cell), and striped (non-stellate cell). The indicated clades were previously defined based on the phylogeny of the group (Roalson et al. 2010Roalson EH, Hinchliff CE, Trevisan R & Silva CRM (2010) Phylogenetic relationships in Eleocharis (Cyperaceae): C4 photosynthesis origins and patterns of diversification in the spikerushes. Systematic Botany 35: 257-271.).
We can conclude that although the species of the genus live in an environment of high plasticity, they developed few variations in the anatomical and micromorphological structures of their diaphragms. None of the characteristics raised are exclusive among the clades, but they contribute to the characterization of the groups and can collaboratively allow for the understanding of phylogenetic relationships in future studies of the genus. The evolution of the raised characteristics demonstrates that the Eleocharis diaphragm is becoming simpler, but the plesiomorphic characteristics are still the most frequent within the group.
Reconstruction of the ancestral state in Eleocharis, by the parsimony method (Mesquite v. 3.03), based on the strict consensus tree by the TNT program (v.1.1); Thickening of the walls of the stem diaphragm cells; black (present), white (absent) and X (not analyzed). The indicated clades were previously defined based on the phylogeny of the group (Roalson et al. 2010).
Acknowledgements
This study was financed in part by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001 - MSc scholarship to RMP; and by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). We thank Dr. Eric H. Roalson (School of Biological Sciences, Washington State University), for making the data for the phylogeny available; to MBM Herbarium employees, for permission to collect samples; and the Center of Electron Microscopy of UFPR, for the use of laboratories and equipment.
Data availability statement
In accordance with Open Science communication practices, the authors inform that all data are available within the manuscript.
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16 Nov 2023
Thickening of the diaphragm cells wall