Fusoid cells in the grass family Poaceae (Poales): a developmental study reveals homologies and suggests new insights into their functional role in young leaves

Fusoid cells in the grass family Poaceae (Poales): a developmental study reveals homologies and... Abstract Background and Aims In mature grass leaf blades as seen in cross-section, oblong cell-like structures have been interpreted most recently as intercellular gas spaces delimited by successive collapsed fusoid cells. These cells have been reported in at least seven of 12 subfamilies of Poaceae and are considered a synapomorphy for the family; however, no developmental work has been performed to verify their meristematic origin or to assess possible homologies within the graminid clade (= Flagellariaceae + [(Joinvilleaceae + Ecdeiocoleaceae) + Poaceae]) or among subfamilies of Poaceae. A developmental study was therefore carried out, including 20 species in three families (Flagellariaceae, Joinvilleaceae and Poaceae), representing the earlier-diverging and derived branches within the graminid clade and Poaceae. Methods Light microscopy was combined with scanning electron microscopy, cryoscanning electron microscopy and transmission electron microscopy to study the development of leaves taken from the shoot apex of young plants. Mature leaf blades also were taken from living or dried plants and the mid-portion was studied. Key Results Developmental results show that, in mature leaf blades as seen in cross-section, one apparent fusoid cell is typically a cavity resulting from the collapse of the initial fusoid cell and its internal divisions, which are herein interpreted as derivative cells with formation of cell plates only. Each cavity is delimited by successive collapsed fusoid cells arranged perpendicularly to the veins. Fusoid cells in all studied Poaceae members originate from the ground meristem, as do the colourless cells in Joinvillea ascendens (Joinvilleaceae). These two types of mesophyll cell have a strongly similar ontogeny, distinguished mainly by the collapse of the fusoid cells in Poaceae, which is not observed in the colourless cells in J. ascendens. Conclusions Within the Poaceae, the meristematic origin of fusoid cells is the same in the early-diverging lineages, BOP clade and Panicoideae, and thus they are homologous within the family. The same topography and meristematic origin suggest that fusoid cells in Poaceae and colourless cells in Joinvilleaceae are homologous. The results also suggest that the role played by the fusoid cells in young grass leaves is related to synthesis and storage of starch granules at early stages of development. BOP clade, early-diverging grass lineages, Flagellariaceae, foliar anatomy, graminid clade, grasses, Joinvilleaceae, mesophyll cells, ontogeny, Panicoideae INTRODUCTION Poales is a diverse group that currently comprises 14 families with morphological, anatomical, embryological and molecular similarities (Stevens, 2001 onwards; Angiosperm Phylogeny Group IV, 2016). There are six clades within this order, of which the Flagellariaceae + [(Joinvilleaceae + Ecdeiocoleaceae) + Poaceae] is recognized as the graminid clade (Linder and Rudall, 2005); these four families share primarily morphological and embryological features but also are robustly supported as a clade based on plastome sequences (Stevens, 2001 onwards; Givnish et al., 2010). The graminid family Poaceae comprises nearly 12 000 species in 12 subfamilies widespread mainly in grasslands and forests all over the world (Grass Phylogeny Working Group II, 2012; Soreng et al., 2015, 2017), diversity for which anatomical studies have often provided useful features that aid in identification and delimitation at various taxonomic levels (e.g. Calderón and Soderstrom, 1973; Zuloaga et al., 1992, 1993; Aliscioni et al., 2003, 2016; Guglieri et al., 2008; Oliveira et al., 2008; Pelegrin et al., 2009; Leandro et al., 2016a), and also often indicate potential synapomorphies for clades (Judziewicz et al., 1999; Grass Phylogeny Working Group, 2001; Clark et al., 2015; Leandro et al., 2016b, 2017). Although knowledge of grass anatomy is constantly growing and there are many contributions in the literature, anatomical work on grasses has concentrated mainly on analyses of leaf-blade cross-sections using light microscopy. Strongly asymmetrically invaginated arm cells, intercostal fibres, Kranz anatomy and bundle spacing are examples of highly systematically informative leaf-blade features as seen in cross-section (e.g. Judziewicz et al., 1999; Grass Phylogeny Working Group, 2001; Viana et al., 2011; Grass Phylogeny Working Group II, 2012; Christin et al., 2013; Clark et al., 2015; Leandro et al., 2016b, 2017). In contrast, the systematic value of fusoid cells, an important leaf-blade anatomical feature for Poaceae, is still controversial in some instances and conflicting findings regarding their taxonomic distribution and functional role(s) in mature leaves have been published (see below). The term ‘fusoid cells’ was originally used by Metcalfe (1956), who studied the leaf-blade anatomy of many bamboo species (see the terminological history in Table 1). Even today, fusoid cells are assumed to be typical of Bambusoideae species, even though these cells are also observed in other subfamilies across the family (Grass Phylogeny Working Group, 2001): (1) the early-diverging lineages (Anomochlooideae, Pharoideae and Puelioideae) (Ellis, 1987—Anomochlooideae and Pharoideae referred to as Bambusoideae in this work; Clark et al., 2000); (2) the BOP clade (Bambusoideae and also in a few members of Oryzoideae and Pooideae) (Tateoka, 1963; Barkworth et al., 2007; Leandro et al., 2016a); and (3) the PACMAD clade (in a few members of Panicoideae) (Watson et al., 1985; Clayton and Renvoize, 1986; Killeen and Clark, 1986; Zuloaga et al., 1992). A summary phylogeny showing the relationships within the family and the occurrence of fusoid cells is provided as supplementary material (Supplementary Data S1). Table 1. Brief terminological history of fusoid cells Reference  Taxon  Anatomical term  Karelstschicoff (1868)  Dendrocalamus strictus  Faltenzellen, ‘folded cells’ (described as many thin-walled cells with different planes overarched)  Brandis (1907)  Bambusoideae  Apparent cavities (although described as cavities, they are recognized as cells)  Arber (1934)  Sasa disticha  Colourless central mesophyll cells  Page (1947)  Streptochaeta spicata  Enlarged mesophyll cells (with regard to the big cells in the mesophyll as seen in cross-section, longitudinal section and paradermal section)  Jacques-Félix (1954)  Bambusoideae  Lacune ou parenchyme différencié (with regard to the anastomosing network of canals that branch and then reunite as before)  Metcalfe (1956)  Bambusoideae  Fusoid cells (to define the fusiform-shaped cells as seen in cross-section)  Metcalfe (1960)  Bambusoideae  Fusoid cells  Wu (1960)  Bambuseae  Translucent fusoid cells (according to Metcalfe (1956), who defined these as fusoid cells)  Jacques-Félix (1962)  Oxytenanthera abyssinica  Cellules claires, ‘clear cells’  Renvoize (1985)  Bambusoideae  Fusoid cells  Killeen and Clark (1986)  Panicum sect. Laxa  Fusoid-like cells similar to cavities that apparently are derived from laterally extended bundle sheaths  Renvoize (1987)  Bambuseae  fusoid cells  Clark (1991)  Bambusoideae  Fusoid cells or spaces formed by their collapse (with regard to the spaces as seen in cross-section and longitudinal section)  Zuloaga et al. (1992)  Panicum subg. Phanophyrum sect. Laxa  Translucent fusoid cells, fusoid cells, and fusoid cavities (used in the same work to refer to the same cell type)  Grass Phylogeny Working Group (2001)  Poaceae  Fusoid cells for early-diverging lineages and Bambusoideae, and fusoid-like cells for Panicoideae  Vega et al. (2016)  Guadua species  Intercellular gas spaces (with regard to fusoid cell collapse and the space left between adjacent fusoid cells)  Wang et al. (2016)  Bambusoideae  Fusoid cells  Reference  Taxon  Anatomical term  Karelstschicoff (1868)  Dendrocalamus strictus  Faltenzellen, ‘folded cells’ (described as many thin-walled cells with different planes overarched)  Brandis (1907)  Bambusoideae  Apparent cavities (although described as cavities, they are recognized as cells)  Arber (1934)  Sasa disticha  Colourless central mesophyll cells  Page (1947)  Streptochaeta spicata  Enlarged mesophyll cells (with regard to the big cells in the mesophyll as seen in cross-section, longitudinal section and paradermal section)  Jacques-Félix (1954)  Bambusoideae  Lacune ou parenchyme différencié (with regard to the anastomosing network of canals that branch and then reunite as before)  Metcalfe (1956)  Bambusoideae  Fusoid cells (to define the fusiform-shaped cells as seen in cross-section)  Metcalfe (1960)  Bambusoideae  Fusoid cells  Wu (1960)  Bambuseae  Translucent fusoid cells (according to Metcalfe (1956), who defined these as fusoid cells)  Jacques-Félix (1962)  Oxytenanthera abyssinica  Cellules claires, ‘clear cells’  Renvoize (1985)  Bambusoideae  Fusoid cells  Killeen and Clark (1986)  Panicum sect. Laxa  Fusoid-like cells similar to cavities that apparently are derived from laterally extended bundle sheaths  Renvoize (1987)  Bambuseae  fusoid cells  Clark (1991)  Bambusoideae  Fusoid cells or spaces formed by their collapse (with regard to the spaces as seen in cross-section and longitudinal section)  Zuloaga et al. (1992)  Panicum subg. Phanophyrum sect. Laxa  Translucent fusoid cells, fusoid cells, and fusoid cavities (used in the same work to refer to the same cell type)  Grass Phylogeny Working Group (2001)  Poaceae  Fusoid cells for early-diverging lineages and Bambusoideae, and fusoid-like cells for Panicoideae  Vega et al. (2016)  Guadua species  Intercellular gas spaces (with regard to fusoid cell collapse and the space left between adjacent fusoid cells)  Wang et al. (2016)  Bambusoideae  Fusoid cells  View Large Table 1. Brief terminological history of fusoid cells Reference  Taxon  Anatomical term  Karelstschicoff (1868)  Dendrocalamus strictus  Faltenzellen, ‘folded cells’ (described as many thin-walled cells with different planes overarched)  Brandis (1907)  Bambusoideae  Apparent cavities (although described as cavities, they are recognized as cells)  Arber (1934)  Sasa disticha  Colourless central mesophyll cells  Page (1947)  Streptochaeta spicata  Enlarged mesophyll cells (with regard to the big cells in the mesophyll as seen in cross-section, longitudinal section and paradermal section)  Jacques-Félix (1954)  Bambusoideae  Lacune ou parenchyme différencié (with regard to the anastomosing network of canals that branch and then reunite as before)  Metcalfe (1956)  Bambusoideae  Fusoid cells (to define the fusiform-shaped cells as seen in cross-section)  Metcalfe (1960)  Bambusoideae  Fusoid cells  Wu (1960)  Bambuseae  Translucent fusoid cells (according to Metcalfe (1956), who defined these as fusoid cells)  Jacques-Félix (1962)  Oxytenanthera abyssinica  Cellules claires, ‘clear cells’  Renvoize (1985)  Bambusoideae  Fusoid cells  Killeen and Clark (1986)  Panicum sect. Laxa  Fusoid-like cells similar to cavities that apparently are derived from laterally extended bundle sheaths  Renvoize (1987)  Bambuseae  fusoid cells  Clark (1991)  Bambusoideae  Fusoid cells or spaces formed by their collapse (with regard to the spaces as seen in cross-section and longitudinal section)  Zuloaga et al. (1992)  Panicum subg. Phanophyrum sect. Laxa  Translucent fusoid cells, fusoid cells, and fusoid cavities (used in the same work to refer to the same cell type)  Grass Phylogeny Working Group (2001)  Poaceae  Fusoid cells for early-diverging lineages and Bambusoideae, and fusoid-like cells for Panicoideae  Vega et al. (2016)  Guadua species  Intercellular gas spaces (with regard to fusoid cell collapse and the space left between adjacent fusoid cells)  Wang et al. (2016)  Bambusoideae  Fusoid cells  Reference  Taxon  Anatomical term  Karelstschicoff (1868)  Dendrocalamus strictus  Faltenzellen, ‘folded cells’ (described as many thin-walled cells with different planes overarched)  Brandis (1907)  Bambusoideae  Apparent cavities (although described as cavities, they are recognized as cells)  Arber (1934)  Sasa disticha  Colourless central mesophyll cells  Page (1947)  Streptochaeta spicata  Enlarged mesophyll cells (with regard to the big cells in the mesophyll as seen in cross-section, longitudinal section and paradermal section)  Jacques-Félix (1954)  Bambusoideae  Lacune ou parenchyme différencié (with regard to the anastomosing network of canals that branch and then reunite as before)  Metcalfe (1956)  Bambusoideae  Fusoid cells (to define the fusiform-shaped cells as seen in cross-section)  Metcalfe (1960)  Bambusoideae  Fusoid cells  Wu (1960)  Bambuseae  Translucent fusoid cells (according to Metcalfe (1956), who defined these as fusoid cells)  Jacques-Félix (1962)  Oxytenanthera abyssinica  Cellules claires, ‘clear cells’  Renvoize (1985)  Bambusoideae  Fusoid cells  Killeen and Clark (1986)  Panicum sect. Laxa  Fusoid-like cells similar to cavities that apparently are derived from laterally extended bundle sheaths  Renvoize (1987)  Bambuseae  fusoid cells  Clark (1991)  Bambusoideae  Fusoid cells or spaces formed by their collapse (with regard to the spaces as seen in cross-section and longitudinal section)  Zuloaga et al. (1992)  Panicum subg. Phanophyrum sect. Laxa  Translucent fusoid cells, fusoid cells, and fusoid cavities (used in the same work to refer to the same cell type)  Grass Phylogeny Working Group (2001)  Poaceae  Fusoid cells for early-diverging lineages and Bambusoideae, and fusoid-like cells for Panicoideae  Vega et al. (2016)  Guadua species  Intercellular gas spaces (with regard to fusoid cell collapse and the space left between adjacent fusoid cells)  Wang et al. (2016)  Bambusoideae  Fusoid cells  View Large Although fusoid cells are recognized as a prominent feature of the early-diverging lineages and Bambusoideae, the role played by these cells in mature leaves has never been satisfactorily explained. Clark (1991) proposed that fusoid cells act as reservoirs for CO2 from photorespiration, a hypothesis later rejected by March and Clark (2011) based on the typically lower rates of photorespiration in shaded environments and the greater development of fusoid cells in shade leaves. Rather, March and Clark (2011) proposed that cavities formed by the collapse of fusoid cells may be related to intra- or intercellular reflectance, which contributes to the absorption and intrafoliar redistribution of light in shaded environments. Vega et al. (2016) proposed that the I-shaped collapsed fusoid cells as seen in longitudinal section may play a structural role, supporting the mature leaf-blade architecture. Vieira et al. (2002) and Wang et al. (2016), in turn, suggested the relationship of fusoid cells to storage, transportation and water balance functions. Wang et al. (2016) also reinforced the potential role of fusoid cells as CO2 reservoirs from photorespiration based on greenhouse experiments under high temperatures. Given their wide occurrence across Poaceae, fusoid cells have been considered an important character in taxonomic descriptions and also in establishing phylogenetic relationships within the family. On the other hand, previous work has shown that these cells are environmentally influenced and thus their occurrence may be facultative within the same species or even within the same sample or individual (e.g. Metcalfe, 1956; Wu, 1962; Pearson et al., 1994; March and Clark, 2011). For example, in bamboos, plants living in sunny habitats often lack these cells, whereas the same species growing in shade always have them (e.g. March and Clark, 2011); in contrast, Oryzeae (Oryzoideae) comprise aquatic or mesic species, which may exhibit fusoid cells, but primarily live in open habitats (Tateoka, 1963; Leandro et al., 2016a). In mature leaf blades as seen in cross-section, the fusoid cells are large, mainly more or less oblong with thin cell walls, and apparently with no content (Grass Phylogeny Working Group, 2001; Kellogg, 2015). This set of features makes these cells visually similar to cavities or air spaces in the mesophyll, consequently generating doubts about whether they are cells or intercellular spaces (Karelstschicoff, 1868; Brandis, 1907). Their cellular nature was confirmed in studies of leaf-blade development in Streptochaeta (Page, 1947) and the bamboo Oxytenanthera abyssinica (Jacques-Félix, 1962), in which the fusoid cells were called ‘enlarged mesophyll cells’ and ‘cellules claires’, respectively (Table 1). A recent study of the development of fusoid cells in two species of Guadua (Bambusoideae) show that these cells differentiate, enlarge and then usually collapse to form intercellular gas spaces as seen in cross-section (Vega et al., 2016; Table 1). These three developmental studies, although addressing aspects of fusoid cell ontogeny, do not completely clarify the origin of these cells or their occurrence or homology across the grass family. Since there are no leaf developmental studies addressing the occurrence of fusoid cells and their homologies among families within the graminid clade, there is no consensus with regard to a number of questions: (1) whether the presence of fusoid cells is in fact a synapomorphy for the Poaceae (Grass Phylogeny Working Group, 2001), considering their occurrence in the early-diverging lineages and the presence of colourless cells similar to fusoid cells in the mesophyll of Joinvilleaceae members; (2) whether fusoid cells were lost multiple times within the BOP clade, considering the absence of fusoid cells in many Oryzoideae and in most Pooideae members (Grass Phylogeny Working Group, 2001); and (3) whether their origin is the same across the entire family – they are called ‘fusoid-like cells’ in the Panicoideae (Killeen and Clark, 1986). Hence, given the importance of fusoid cells for grass systematics and phylogeny, the primary aims of this work were: (1) to study leaf-blade development within Poaceae focusing on the development of fusoid cells in order to verify their meristematic origin and putative homologies; and (2) to determine whether the colourless cells of the mesophyll similar to fusoid cells observed in Joinvilleaceae have the same origin as in the Poaceae. To that end, we studied the development of 20 species among Poaceae, Joinvilleaceae and Flagellariaceae (the graminid clade). MATERIALS AND METHODS Sampling For the developmental work the following taxa were studied: (1) Joinvillea ascendens (Joinvilleaceae); and (2) several Poaceae members known to have fusoid cells in six subfamilies: the two earliest-diverging lineages (Anomochlooideae and Pharoideae), the BOP clade (Bambusoideae, Oryzoideae and Pooideae) and the PACMAD clade (Panicoideae). Since we examined leaf-blade development in Joinvilleaceae in order to compare it with Poaceae, we also included Flagellaria indica (Flagellariaceae), placed within the graminid clade and sister to [(Joinvilleaceae + Ecdeiocoleaceae) + Poaceae] (Stevens, 2001 onwards; Linder and Rudall, 2005). To define homologies properly we also included selected species across the grass family in which fusoid cells are lacking. Living plants were sampled in their natural habitat or from the R. W. Pohl Conservatory at Iowa State University, USA. We were not able to collect living plants to represent the subfamily Puelioideae (from Tropical Africa), but we included anatomical results from mature dried leaves of Guaduella oblonga (Puelioideae) and also from other Poaceae species in order to provide a broad perspective. All species included in this work, sampling method, purpose of study and vouchers are provided in Table 2. Note that although there are differences in mesophyll arrangement and vascular bundle spacing between C3 and C4 species (Dengler et al., 1994; Ueno et al., 2006), Setaria scabrifolia (C4) was considered in this study since our main aim was only to compare the development of ground meristem cells into types of mesophyll cells, especially fusoid cells. Table 2. List of studied species, occurrence of fusoid cells, vouchers and types of microscopy and analysis. Taxon  Fusoid cells  Voucher  Analysis performed        LM  EM        DV  AN  CS  T  Flagellariaceae   Flagellaria indica L.  –  Clark & Zhang 1305 (ISC)  X  X      Joinvilleaceae   Joinvillea ascendens Gaudich. ex Brongn. & Gris  –  Clark & Attigala 1714 (ISC)  X  X    X  Poaceae   Anomochlooideae    Streptochaeta spicata Schrad. ex Nees  +  Clark & Lewis 1642 (ISC)  X  X    X   Pharoideae    Pharus latifolius L.  +  Klahs 1250 (ISC)  X  X    X   Puelioideae    Guaduella oblonga Hutchinson ex W. D. Clayton  +  Baldwin 6704 (ISC)    X    X   Oryzoideae    Ehrharta erecta Lam.  –  Clark & Gallaher (ISC)  X  X        Luziola spruceana Benth. ex Döll  +  Moreira 249 (CGMS)    X        Oryza latifolia Desv.  +  Guglieri-Caporal 3172 (CGMS)    X       Bambusoideae    Raddia brasiliensis Bertol.  +  Clark & Attigala 1713 (ISC)  X  X        Parodiolyra micrantha (Kunth) Davidse & Zuloaga  +  Shirasuna 2863 (SP)    X        Aulonemia aristulata (Döll) McClure  +  Shirasuna 2860 (SP)  X  X    X    Chusquea attenuata (Döll) L.G. Clark  +  Campos et al. s.n. (ISC)  X          Guadua angustifolia Kunth  +  Clark 1095 (ISC)      X      Otatea rzedowskiorum Ruiz-Sanchez  +  Clark s.n. (ISC)  X  X       Pooideae    Brachyelytrum erectum (Schreb.) P. Beauv.  –  Clark & Dixon 1669 (ISC)  X  X        Diarrhena obovata (Gleason) Brandenburg  –  Clark & Dixon 1667 (ISC)  X  X        Glyceria striata (Lam.) Hitchc.  +  Clark & Dixon 1671 (ISC)  X  X       Panicoideae    Centotheca lappacea (L.) Desv.  –  Sanchez-Ken s.n. (ISC)  X  X        Rugoloa pilosa (Sw.) Zuloaga  +  Leandro 161 (CGMS)  X  X        Setaria scabrifolia (Nees) Kunth  –  Leandro 160 (CGMS)  X  X      Taxon  Fusoid cells  Voucher  Analysis performed        LM  EM        DV  AN  CS  T  Flagellariaceae   Flagellaria indica L.  –  Clark & Zhang 1305 (ISC)  X  X      Joinvilleaceae   Joinvillea ascendens Gaudich. ex Brongn. & Gris  –  Clark & Attigala 1714 (ISC)  X  X    X  Poaceae   Anomochlooideae    Streptochaeta spicata Schrad. ex Nees  +  Clark & Lewis 1642 (ISC)  X  X    X   Pharoideae    Pharus latifolius L.  +  Klahs 1250 (ISC)  X  X    X   Puelioideae    Guaduella oblonga Hutchinson ex W. D. Clayton  +  Baldwin 6704 (ISC)    X    X   Oryzoideae    Ehrharta erecta Lam.  –  Clark & Gallaher (ISC)  X  X        Luziola spruceana Benth. ex Döll  +  Moreira 249 (CGMS)    X        Oryza latifolia Desv.  +  Guglieri-Caporal 3172 (CGMS)    X       Bambusoideae    Raddia brasiliensis Bertol.  +  Clark & Attigala 1713 (ISC)  X  X        Parodiolyra micrantha (Kunth) Davidse & Zuloaga  +  Shirasuna 2863 (SP)    X        Aulonemia aristulata (Döll) McClure  +  Shirasuna 2860 (SP)  X  X    X    Chusquea attenuata (Döll) L.G. Clark  +  Campos et al. s.n. (ISC)  X          Guadua angustifolia Kunth  +  Clark 1095 (ISC)      X      Otatea rzedowskiorum Ruiz-Sanchez  +  Clark s.n. (ISC)  X  X       Pooideae    Brachyelytrum erectum (Schreb.) P. Beauv.  –  Clark & Dixon 1669 (ISC)  X  X        Diarrhena obovata (Gleason) Brandenburg  –  Clark & Dixon 1667 (ISC)  X  X        Glyceria striata (Lam.) Hitchc.  +  Clark & Dixon 1671 (ISC)  X  X       Panicoideae    Centotheca lappacea (L.) Desv.  –  Sanchez-Ken s.n. (ISC)  X  X        Rugoloa pilosa (Sw.) Zuloaga  +  Leandro 161 (CGMS)  X  X        Setaria scabrifolia (Nees) Kunth  –  Leandro 160 (CGMS)  X  X      CGMS, Herbarium of Universidade Federal de Mato Grosso do Sul; ISC, Ada Hayden Herbarium of Iowa State University; SP, Herbarium of the Instituto de Botânica de São Paulo. AN, anatomical work (mature leaves); DV, developmental work (ontogeny); LM, light microscopy; EM, electron microscopy; CS, cryoscanning electron microscopy; T, transmission electron microscopy. +, presence; –, absence. View Large Table 2. List of studied species, occurrence of fusoid cells, vouchers and types of microscopy and analysis. Taxon  Fusoid cells  Voucher  Analysis performed        LM  EM        DV  AN  CS  T  Flagellariaceae   Flagellaria indica L.  –  Clark & Zhang 1305 (ISC)  X  X      Joinvilleaceae   Joinvillea ascendens Gaudich. ex Brongn. & Gris  –  Clark & Attigala 1714 (ISC)  X  X    X  Poaceae   Anomochlooideae    Streptochaeta spicata Schrad. ex Nees  +  Clark & Lewis 1642 (ISC)  X  X    X   Pharoideae    Pharus latifolius L.  +  Klahs 1250 (ISC)  X  X    X   Puelioideae    Guaduella oblonga Hutchinson ex W. D. Clayton  +  Baldwin 6704 (ISC)    X    X   Oryzoideae    Ehrharta erecta Lam.  –  Clark & Gallaher (ISC)  X  X        Luziola spruceana Benth. ex Döll  +  Moreira 249 (CGMS)    X        Oryza latifolia Desv.  +  Guglieri-Caporal 3172 (CGMS)    X       Bambusoideae    Raddia brasiliensis Bertol.  +  Clark & Attigala 1713 (ISC)  X  X        Parodiolyra micrantha (Kunth) Davidse & Zuloaga  +  Shirasuna 2863 (SP)    X        Aulonemia aristulata (Döll) McClure  +  Shirasuna 2860 (SP)  X  X    X    Chusquea attenuata (Döll) L.G. Clark  +  Campos et al. s.n. (ISC)  X          Guadua angustifolia Kunth  +  Clark 1095 (ISC)      X      Otatea rzedowskiorum Ruiz-Sanchez  +  Clark s.n. (ISC)  X  X       Pooideae    Brachyelytrum erectum (Schreb.) P. Beauv.  –  Clark & Dixon 1669 (ISC)  X  X        Diarrhena obovata (Gleason) Brandenburg  –  Clark & Dixon 1667 (ISC)  X  X        Glyceria striata (Lam.) Hitchc.  +  Clark & Dixon 1671 (ISC)  X  X       Panicoideae    Centotheca lappacea (L.) Desv.  –  Sanchez-Ken s.n. (ISC)  X  X        Rugoloa pilosa (Sw.) Zuloaga  +  Leandro 161 (CGMS)  X  X        Setaria scabrifolia (Nees) Kunth  –  Leandro 160 (CGMS)  X  X      Taxon  Fusoid cells  Voucher  Analysis performed        LM  EM        DV  AN  CS  T  Flagellariaceae   Flagellaria indica L.  –  Clark & Zhang 1305 (ISC)  X  X      Joinvilleaceae   Joinvillea ascendens Gaudich. ex Brongn. & Gris  –  Clark & Attigala 1714 (ISC)  X  X    X  Poaceae   Anomochlooideae    Streptochaeta spicata Schrad. ex Nees  +  Clark & Lewis 1642 (ISC)  X  X    X   Pharoideae    Pharus latifolius L.  +  Klahs 1250 (ISC)  X  X    X   Puelioideae    Guaduella oblonga Hutchinson ex W. D. Clayton  +  Baldwin 6704 (ISC)    X    X   Oryzoideae    Ehrharta erecta Lam.  –  Clark & Gallaher (ISC)  X  X        Luziola spruceana Benth. ex Döll  +  Moreira 249 (CGMS)    X        Oryza latifolia Desv.  +  Guglieri-Caporal 3172 (CGMS)    X       Bambusoideae    Raddia brasiliensis Bertol.  +  Clark & Attigala 1713 (ISC)  X  X        Parodiolyra micrantha (Kunth) Davidse & Zuloaga  +  Shirasuna 2863 (SP)    X        Aulonemia aristulata (Döll) McClure  +  Shirasuna 2860 (SP)  X  X    X    Chusquea attenuata (Döll) L.G. Clark  +  Campos et al. s.n. (ISC)  X          Guadua angustifolia Kunth  +  Clark 1095 (ISC)      X      Otatea rzedowskiorum Ruiz-Sanchez  +  Clark s.n. (ISC)  X  X       Pooideae    Brachyelytrum erectum (Schreb.) P. Beauv.  –  Clark & Dixon 1669 (ISC)  X  X        Diarrhena obovata (Gleason) Brandenburg  –  Clark & Dixon 1667 (ISC)  X  X        Glyceria striata (Lam.) Hitchc.  +  Clark & Dixon 1671 (ISC)  X  X       Panicoideae    Centotheca lappacea (L.) Desv.  –  Sanchez-Ken s.n. (ISC)  X  X        Rugoloa pilosa (Sw.) Zuloaga  +  Leandro 161 (CGMS)  X  X        Setaria scabrifolia (Nees) Kunth  –  Leandro 160 (CGMS)  X  X      CGMS, Herbarium of Universidade Federal de Mato Grosso do Sul; ISC, Ada Hayden Herbarium of Iowa State University; SP, Herbarium of the Instituto de Botânica de São Paulo. AN, anatomical work (mature leaves); DV, developmental work (ontogeny); LM, light microscopy; EM, electron microscopy; CS, cryoscanning electron microscopy; T, transmission electron microscopy. +, presence; –, absence. View Large Developmental study (young leaves) Light microscopy (LM). Leaves were taken from the shoot apex of young plants, fixed in FAA50 (formaldehyde–acetic acid–ethanol) for 48 h (Johansen, 1940) and then stored in 70 % ethanol. Each sample was dehydrated through a graded n-butyl series and embedded in glycol methacrylate (Leica Historesin Embedding Kit) following conventional methods. Cross-sections were obtained with a rotary microtome (Leica RM 2145 or Spencer 820) and stained with toluidine blue (Feder and O’Brien, 1968) to visualize cellulosic cell walls plus periodic acid–Schiff (PAS) to provide better contrast. Permanent slides were mounted with Entellan (Merck, Germany) or Permount (Fisher, USA). Transmission electron microscopy (TEM). Small leaf-blade pieces (1 mm diameter) from the shoot apex of young plants were fixed with 2 % paraformaldehyde/2 % glutaraldehyde in a 0.1 m cacodylate buffer at 4 ºC for 48 h (Horner, 2012). The pieces were buffer-washed three times for a total of 1 h, dehydrated through an ethanol series, and embedded in Spurr’s resin (Spurr Low-Viscosity Embedding Kit). Ultra-thin sections were cut with a diamond knife then placed on copper grids and post-stained with lead and uranium. Anatomical study (mature leaf blades) Light microscopy. Mature leaf blades were taken from living or dried plants and sampled from the middle portion of the blade. For living plants, each sample was fixed in FAA50 for 48 h (Johansen, 1940) and stored in 70 % ethanol. Due to the amount of sclerified and silicified tissues, sections were made by using two distinct methods: (1) leaf-blade pieces (0.5 cm2) were embedded in polyethylene glycol 1500 solution and kept in an incubator at 60 ºC for 15 d, dried and then sectioned (adapted from Richter, 1985); or (2) samples were treated with 30 % hydrofluoric acid for 72 h, dehydrated through a graded n-ethanol series, embedded in paraffin and then sectioned. For both methods, sections were made using a Leica RM 2145 or Spencer 820 rotary microtome and then stained with epoxy tissue stain (Spurlock et al., 1966). Permanent slides were mounted with Entellan (Merck, Germany). Also, pieces of ~0.5 cm2 from the middle portion of dried leaf blades of Chusquea attenuata were removed for analysis of fusoid cells through the depth of the leaf blade. These pieces were hydrated through a graded series of ethyl alcohol (50, 25 and 10 %) and then soaked in 1:1/dH20 (deionized water) until the samples were translucent. Samples were rinsed with dH20, dehydrated through a graded series of ethyl alcohol (25, 50 and 70 %) and then stained with safranin and fast green (Johansen, 1940). Stained samples were treated with ethyl alcohol (95 and 100 %), xylene and then xylene and Permount (1:1). Permanent slides were mounted with Permount (Fisher, USA). Cryoscanning electron microscopy (CSEM). Pieces of ~ 1 cm2 from the middle portion of living leaf blades of Guadua angustifolia were vertically attached to a cylindrical sample holder (stub) with cryoglue (Tissue-Tec, Scigen Scientific, USA) and then frozen in a bath of liquid nitrogen with vacuum applied. These samples were placed in a cryopreparation chamber (Quorum Cryo System PPT 3010-T) and then cryofractured with a semirotary cryoknife to expose the mesophyll tissues in cross-section. Description, observation, images and tridimensional reconstruction Descriptions and illustrations mainly concentrated on the development of fusoid cells and the terminology primarily followed Ellis (1976, 1979). In order to better present the results, the progression of leaf development was divided into four stages. For LM analyses, photomicrographs were obtained with a Zeiss Axio Observer microscope using ZEN 2.0 blue software or with a Leica DM4000B microscope using the Leica Application Suite LASV4.0. Scanning/transmission electron microscope images (STEM) were captured with a JEOL 2100 or FEI Tecnai™ Spirit TEM. In order to build a paradermal scan with tridimensional reconstruction showing the fusoid cells through the depth of the leaf blade, each 0.5-μm-thick section of a leaf-blade clearing of C. attenuata was photographed along the Z-axis to create a stack of eight to ten 0.5-μm-thick optical sections. Z-stacks from each section were aligned and modelled also using ZEN 2.0 blue. The analyses herein described were performed in the Departamento de Botânica at UNESP-Rio Claro, Brazil, in the Centro de Microscopia Eletrônica (CME) at UNESP-Botucatu, Brazil, and in the Roy J. Carver High Resolution Microscopy Facility (HRMF) at Iowa State University, USA. RESULTS The following descriptions include four stages of leaf development with an emphasis on the fusoid cells (Figs 1A–F, 2A–J, 3A–G and 4A–Z, Poaceae). Images for the development of leaves with colourless cells (Fig. 5A–E, Joinvilleaceae) and with no fusoid cells (Fig. 6A–I, Poaceae and Flagellariaceae) are included, as well as a video showing a tridimensional reconstruction of mature fusoid cells (Supplementary Data Video). The text and figures mix descriptions of several studied species, but a full developmental series of Aulonemia aristulata, a Bambusoideae member that exhibits a typical development of fusoid cells, is provided as supplementary material (Supplementary Data S2). Fig. 1. View largeDownload slide Cross-sections of leaf blades (LM) at early stages of development with emphasis on the development of fusoid cells in Poaceae species. (A, B, D, E) Aulonemia aristulata (Bambusoideae); (C, F) Rugoloa pilosa (Panicoideae). Red arrows show cell divisions, blue coloration highlights procambial strands (pr) and pink coloration highlights the differentiation of ground meristem (gm) into fusoid cells. (A) Early procambial strand of the midvein, protoderm (pt) and ground meristem. (B) Midvein and initial development of minor procambial strands. (C) Minor procambial strands are formed between pre-existing major ones. (D) Major and minor procambial strands. (E) Subsequent differentiation of procambial strands into vascular bundles and ground meristem cells into fusoid cells. (F) Differentiation of protoderm and procambium, as well as ground meristem cells with fusoid cells readily distinguishable. OBS: For additional information, see supplementary note provided in the Supplementary Data S2. Scale bars = 15 µm. Fig. 1. View largeDownload slide Cross-sections of leaf blades (LM) at early stages of development with emphasis on the development of fusoid cells in Poaceae species. (A, B, D, E) Aulonemia aristulata (Bambusoideae); (C, F) Rugoloa pilosa (Panicoideae). Red arrows show cell divisions, blue coloration highlights procambial strands (pr) and pink coloration highlights the differentiation of ground meristem (gm) into fusoid cells. (A) Early procambial strand of the midvein, protoderm (pt) and ground meristem. (B) Midvein and initial development of minor procambial strands. (C) Minor procambial strands are formed between pre-existing major ones. (D) Major and minor procambial strands. (E) Subsequent differentiation of procambial strands into vascular bundles and ground meristem cells into fusoid cells. (F) Differentiation of protoderm and procambium, as well as ground meristem cells with fusoid cells readily distinguishable. OBS: For additional information, see supplementary note provided in the Supplementary Data S2. Scale bars = 15 µm. Fig. 2. View largeDownload slide Cross-sections of leaf blades (LM) at early stages of development with emphasis on the development of fusoid cells in Poaceae species. (A) Rugoloa pilosa (Panicoideae); (B) Aulonemia aristulata (Bambusoideae); (C, D, E, H) Otatea rzedowskiorum (Bambusoideae); (F, G, I, J) Pharus latifolius (Pharoideae). Red arrows show cell divisions, pink coloration highlights the differentiation of ground meristem into fusoid cells, and asterisks show the initial cavities formed from the degradation of the middle lamella and gradual collapse of fusoid cells. (A) Adaxial (ad) and abaxial (ab) epidermis and vascular bundles (vb) differentiated (except the bulliform cells), and differentiation of ground meristem cells with fusoid cells readily distinguishable. (B) Several fusoid cells adjacent to the vascular bundles and inset showing a young fusoid cell with content. (C) Initial fusoid cell giving rise to a derivative fusoid cell (internal division within an initial fusoid cell). (D) Subsequent division from initial and derivative fusoid cells (internal divisions within an initial fusoid cell). (E) Vascular bundle between one (left) and three (right) fusoid cells, with nuclei visible in the fusoid cells. (F) Degradation of the middle lamella between fusoid and chlorenchyma cells. (G, H) Fusoid cell divisions accompanying vascular bundle maturation. Note the fully differentiated vascular tissue and enlarged bundle sheath cells in (G). (I) Degradation of the middle lamella between fusoid and chlorenchyma cells following bundle maturation. (J) Fusoid cell stretched and flattened along the lateral axis as a result of the degradation of the middle lamella and leaf blade enlargement. OBS: For additional information, see Supplementary Data S2. Scale bars (C, D, E, H) = 10 µm; (A) =15 µm; (I) = 30 µm; (F, G, J) = 40 µm; (B, inset) = 50 µm. Fig. 2. View largeDownload slide Cross-sections of leaf blades (LM) at early stages of development with emphasis on the development of fusoid cells in Poaceae species. (A) Rugoloa pilosa (Panicoideae); (B) Aulonemia aristulata (Bambusoideae); (C, D, E, H) Otatea rzedowskiorum (Bambusoideae); (F, G, I, J) Pharus latifolius (Pharoideae). Red arrows show cell divisions, pink coloration highlights the differentiation of ground meristem into fusoid cells, and asterisks show the initial cavities formed from the degradation of the middle lamella and gradual collapse of fusoid cells. (A) Adaxial (ad) and abaxial (ab) epidermis and vascular bundles (vb) differentiated (except the bulliform cells), and differentiation of ground meristem cells with fusoid cells readily distinguishable. (B) Several fusoid cells adjacent to the vascular bundles and inset showing a young fusoid cell with content. (C) Initial fusoid cell giving rise to a derivative fusoid cell (internal division within an initial fusoid cell). (D) Subsequent division from initial and derivative fusoid cells (internal divisions within an initial fusoid cell). (E) Vascular bundle between one (left) and three (right) fusoid cells, with nuclei visible in the fusoid cells. (F) Degradation of the middle lamella between fusoid and chlorenchyma cells. (G, H) Fusoid cell divisions accompanying vascular bundle maturation. Note the fully differentiated vascular tissue and enlarged bundle sheath cells in (G). (I) Degradation of the middle lamella between fusoid and chlorenchyma cells following bundle maturation. (J) Fusoid cell stretched and flattened along the lateral axis as a result of the degradation of the middle lamella and leaf blade enlargement. OBS: For additional information, see Supplementary Data S2. Scale bars (C, D, E, H) = 10 µm; (A) =15 µm; (I) = 30 µm; (F, G, J) = 40 µm; (B, inset) = 50 µm. Fig. 3. View largeDownload slide Cross-sections of leaf blades (TEM) at later stages of development with emphasis on the development of fusoid cells in Poaceae. (A–D) Parodiolyra micrantha (Bambusoideae); (E, F) Streptochaeta spicata (Anomochlooideae); (G) Pharus latifolius (Pharoideae). ad, adaxial epidermis; ab, abaxial epidermis; cc, chlorenchyma cells. White arrows show starch granules, red arrows show cell divisions and asterisks show the initial cavities formed from the degradation of the middle lamella. (A) Initial and the first derivative fusoid cells (internal division) showing nuclei and cytoplasmic content, large vacuoles and formation of a cell plate. (B) Fusoid cell showing an internal division, with initial formation of cell plate. (C) At least four derivative fusoid cells, with a large vacuole each, all within an initial fusoid cell; showing three nucleoli in the one visible nucleus. (D) Detail of a fusoid cell showing a cell plate, vacuoles and membranes from degraded organelles. (E, F) Fusoid cells with starch granules and initial degradation of the middle lamella. (G) Detail of starch granules in the fusoid cell. Scale bars: (G, H) = 1 µm; (B, I) = 3 µm; (D) = 3.5 µm; (C) = 4 µm; (A) = 5 µm. Fig. 3. View largeDownload slide Cross-sections of leaf blades (TEM) at later stages of development with emphasis on the development of fusoid cells in Poaceae. (A–D) Parodiolyra micrantha (Bambusoideae); (E, F) Streptochaeta spicata (Anomochlooideae); (G) Pharus latifolius (Pharoideae). ad, adaxial epidermis; ab, abaxial epidermis; cc, chlorenchyma cells. White arrows show starch granules, red arrows show cell divisions and asterisks show the initial cavities formed from the degradation of the middle lamella. (A) Initial and the first derivative fusoid cells (internal division) showing nuclei and cytoplasmic content, large vacuoles and formation of a cell plate. (B) Fusoid cell showing an internal division, with initial formation of cell plate. (C) At least four derivative fusoid cells, with a large vacuole each, all within an initial fusoid cell; showing three nucleoli in the one visible nucleus. (D) Detail of a fusoid cell showing a cell plate, vacuoles and membranes from degraded organelles. (E, F) Fusoid cells with starch granules and initial degradation of the middle lamella. (G) Detail of starch granules in the fusoid cell. Scale bars: (G, H) = 1 µm; (B, I) = 3 µm; (D) = 3.5 µm; (C) = 4 µm; (A) = 5 µm. Fig. 4. View largeDownload slide Mature leaf blades with emphasis on fusoid cells in Poaceae. LM (A–X) and CSEM (Y, Z). Cross-sections (A–J) show cavities (asterisks) resulting from the collapse of one to several fusoid cells (arrows). Longitudinal sections (K–R), paradermal sections (S–X) and cross-sections (Y–Z) show that each cavity is delimited by successive collapsed (rarely non-collapsed) fusoid cells arranged perpendicular to the veins along the lateral axis and along the proximo-distal axis of the entire lamina. (A, K, S) Streptochaeta spicata (Anomochlooideae). (B, L, M, N) Pharus latifolius (Pharoideae). (C, U) Guaduella oblonga (Puelioideae). (D) Luziola spruceana (Oryzoideae). (E, O) Oryza latifolia (Oryzoideae). (F) Parodiolyra micrantha (Bambusoideae). (G, Q, W) Aulonemia aristulata (Bambusoideae). (H, V) Otatea rzedowskiorum (Bambusoideae). (I) Glyceria striata (Pooideae). (J, R, X) Rugoloa pilosa (Panicoideae). (P) Raddia brasiliensis (Bambusoideae). (Y, Z) Guadua angustifolia (Bambusoideae). Scale bars: (Y, Z) = 6 µm; (W) = 12 µm; (P, R, X) = 25 µm; (M, N) = 30 µm; (K, L, O, Q, S, V) = 40 µm; (A–C, E–H) = 50 µm; (D) = 60 µm; (I, U) = 90 µm; (J) = 100 µm; (T) = 150 µm. Fig. 4. View largeDownload slide Mature leaf blades with emphasis on fusoid cells in Poaceae. LM (A–X) and CSEM (Y, Z). Cross-sections (A–J) show cavities (asterisks) resulting from the collapse of one to several fusoid cells (arrows). Longitudinal sections (K–R), paradermal sections (S–X) and cross-sections (Y–Z) show that each cavity is delimited by successive collapsed (rarely non-collapsed) fusoid cells arranged perpendicular to the veins along the lateral axis and along the proximo-distal axis of the entire lamina. (A, K, S) Streptochaeta spicata (Anomochlooideae). (B, L, M, N) Pharus latifolius (Pharoideae). (C, U) Guaduella oblonga (Puelioideae). (D) Luziola spruceana (Oryzoideae). (E, O) Oryza latifolia (Oryzoideae). (F) Parodiolyra micrantha (Bambusoideae). (G, Q, W) Aulonemia aristulata (Bambusoideae). (H, V) Otatea rzedowskiorum (Bambusoideae). (I) Glyceria striata (Pooideae). (J, R, X) Rugoloa pilosa (Panicoideae). (P) Raddia brasiliensis (Bambusoideae). (Y, Z) Guadua angustifolia (Bambusoideae). Scale bars: (Y, Z) = 6 µm; (W) = 12 µm; (P, R, X) = 25 µm; (M, N) = 30 µm; (K, L, O, Q, S, V) = 40 µm; (A–C, E–H) = 50 µm; (D) = 60 µm; (I, U) = 90 µm; (J) = 100 µm; (T) = 150 µm. Fig. 5. View largeDownload slide Cross-sections of Joinvillea ascendens (Joinvilleaceae) leaf blades (LM) showing the developmental stages of colourless cells (blue arrows). ab, abaxial; ad, adaxial; cc, chlorenchyma cells; vb, vascular bundle. Early stages of development (A–C) and maturity (D, E). (A, B, C) Subsequent developmental stages of colourless cells. (D) Major vascular bundle and chlorenchyma cells completely differentiated and colourless cells showing intact cell walls. (E) Colourless cells, always non-collapsed. OBS. The rosette-like inclusion observed in one of the colourless cells in (D) is a technical artefact caused by embedding and staining methods. Scale bars: (B–D) = 20 µm; (A) = 25 µm (A); (E) = 40 µm. Fig. 5. View largeDownload slide Cross-sections of Joinvillea ascendens (Joinvilleaceae) leaf blades (LM) showing the developmental stages of colourless cells (blue arrows). ab, abaxial; ad, adaxial; cc, chlorenchyma cells; vb, vascular bundle. Early stages of development (A–C) and maturity (D, E). (A, B, C) Subsequent developmental stages of colourless cells. (D) Major vascular bundle and chlorenchyma cells completely differentiated and colourless cells showing intact cell walls. (E) Colourless cells, always non-collapsed. OBS. The rosette-like inclusion observed in one of the colourless cells in (D) is a technical artefact caused by embedding and staining methods. Scale bars: (B–D) = 20 µm; (A) = 25 µm (A); (E) = 40 µm. Fig. 6. View largeDownload slide Cross-sections of leaf blades (LM) with no fusoid cells at early stages of development (A–D) and at maturity (E–I). (A–F) Setaria scabrifolia (Panicoideae). (G) Brachyelytrum erectum (Pooideae). (H) Centotheca lappacea (Panicoideae). (I) Flagellaria indica (Flagellariaceae). ad, adaxial epidermis; ab, abaxial epidermis; cc, chlorenchyma cells; vb, vascular bundles. Blue coloration highlights procambial strands (pr). (A) Early procambial strand of the midvein, protoderm (pt) and ground meristem (gm). (B) Midvein and initial development of minor procambial strands. (C) Subsequent development of procambial strands. (D) Major and minor procambial strands and initial differentiation of ground meristem cells into chlorenchyma cells. (E–I) Major and minor vascular bundles completely differentiated, as well as the chlorenchyma cells. OBS: For additional information, see supplementary note provided in the Supplementary Data S2. Scale bars: (A–D) = 13 µm; (F, H) = 40 µm; (E, I) = 50 µm; (G) = 100 µm. Fig. 6. View largeDownload slide Cross-sections of leaf blades (LM) with no fusoid cells at early stages of development (A–D) and at maturity (E–I). (A–F) Setaria scabrifolia (Panicoideae). (G) Brachyelytrum erectum (Pooideae). (H) Centotheca lappacea (Panicoideae). (I) Flagellaria indica (Flagellariaceae). ad, adaxial epidermis; ab, abaxial epidermis; cc, chlorenchyma cells; vb, vascular bundles. Blue coloration highlights procambial strands (pr). (A) Early procambial strand of the midvein, protoderm (pt) and ground meristem (gm). (B) Midvein and initial development of minor procambial strands. (C) Subsequent development of procambial strands. (D) Major and minor procambial strands and initial differentiation of ground meristem cells into chlorenchyma cells. (E–I) Major and minor vascular bundles completely differentiated, as well as the chlorenchyma cells. OBS: For additional information, see supplementary note provided in the Supplementary Data S2. Scale bars: (A–D) = 13 µm; (F, H) = 40 µm; (E, I) = 50 µm; (G) = 100 µm. Stage 1: Meristems and initial differentiation of cells into dermal and vascular tissues (LM) The initial stage of leaf-blade development is the same in all studied species and occurs from the base towards the apex (acropetally) and from the centre towards the margins (centrifugally) (e.g. Figs 1A–F and 6A–D). The differentiation of tissues may occur simultaneously, but often the protoderm (outermost meristematic layer) is the first meristem to become differentiated, followed by the procambium (innermost meristematic cells, usually with a large nucleus) and the ground meristem (all the remaining meristematic cells, between protoderm and procambium) (e.g. Figs 1A–F and 6A–D). Protodermal cells start anticlinal divisions to form a single stratum of epidermal cells (e.g. Fig. 1A, B). Simultaneously, procambial cells start anticlinal, periclinal and tangential divisions to give rise to vascular elements (e.g. Fig. 1A–C). Considering the centrifugal differentiation of tissues, the first major procambial strand will form the midvein (e.g. Fig. 1A), and then additional veins are formed by adjacent and also major procambial strands (e.g. Fig. 1B–F). Minor procambial strands are formed between pre-existing major ones as a result of leaf-blade enlargement (e.g. Fig. 1E). Mesophyll precursor cells from the ground meristem are mainly isodiametric and still undifferentiated at this point (e.g. Figs 1A–F and 6A, B), although some ground meristem cells are beginning their differentiation into mesophyll cells, in which cells below the epidermis and adjacent to vascular bundles start to divide mainly in the anticlinal and periclinal planes (e.g. Figs 1D, E and 6B). Some enlargement of the mesophyll cells destined to become fusoid cells may be observed at this stage (e.g. Fig. 1D–F). Stage 2: Ongoing differentiation of dermal, vascular tissues and ground meristem cells (LM) At this stage, the epidermis is completely differentiated (except the bulliform cells, which are large, thin-walled epidermal cells that occur in groups in the intercostal zone of the adaxial epidermis). The midvein and lateral vascular bundles are readily distinguishable, although the maturation of the conducting elements is still in progress (e.g. Fig. 2A–C). According to their position in the mesophyll, cells from the ground meristem will give rise to bundle sheath extensions. Ultimately comprising sclerenchymatous or parenchymatous cells, bundle sheath extensions are formed above and below at least the major vascular bundles, whereas isolated sclerenchymatous cells are mainly formed at the margins. Parenchyma cells that start to become heavily sclerified are observed adjacent to the bulliform cells and adjacent to the abaxial epidermis directly beneath the clusters of bulliform cells in A. aristulata (Poaceae, Bambusoideae) (Fig. 4G, mature leaf). Other mesophyll parenchymatous cells are formed primarily between vascular bundles and can be of six types according to the anatomical path of each species: (1) peg cells – thin-walled photosynthetic cells recognized by forming numerous symmetrical ‘arms’, irregularly spaced; (2) colourless cells – thin-walled cells adjacent to vascular bundles; (3) arm cells – thin-walled photosynthetic cells that exhibit asymmetrical cell wall invaginations (lobes) extending to different depths; (4) rosette cells – thin-walled photosynthetic cells that exhibit shallow cell-wall invaginations (lobes) around their periphery; (5) fusoid cells; and (6) irregular-shaped cells – thin-walled photosynthetic cells with no characteristic shape. Parenchymatous cells differentiate mainly into peg cells in F. indica (Flagellariaceae); irregular-shaped cells and colourless cells in J. ascendens (Joinvilleaceae); irregular-shaped or arm cells and fusoid cells in Streptochaeta spicata (Poaceae, Anomochlooideae) and Pharus latifolius (Poaceae, Pharoideae); irregular-shaped cells in Brachyelytrum erectum, Diarrhena obovata (Poaceae, Pooideae), Centotheca lappacea, Rugoloa pilosa and S. scabrifolia (Poaceae, Panicoideae), and irregular-shaped cells and fusoid cells in Glyceria striata (Poaceae, Pooideae); rosette cells in Ehrharta erecta (Poaceae, Oryzoideae); and arm cells or rosette cells and fusoid cells in A. aristulata, C. attenuata, Otatea rzedowskiorum and Raddia brasiliensis (Poaceae, Bambusoideae). These cell types exhibit contents visible at this stage and will form the chlorenchyma tissue (e.g. Fig. 2A–J). Irregular-shaped cells usually become arranged in a variable number of layers parallel to the epidermis, but in R. pilosa (Fig. 2A) and S. scabrifolia (Fig. 6D, E) they become radiate and surround the vascular bundles. In the members of Bambusoideae herein studied, arm cells usually become arranged into one or two layers of cells below the adaxial epidermis and rosette cells in just one layer above the abaxial epidermis (e.g. Fig. 2B), whereas in E. erecta (Oryzoideae) the number of layers of rosette cells is usually variable (not shown). During the differentiation of the chlorenchyma cells, the cells that occupy the central portion of the mesophyll, adjacent to each side of the vascular bundle, start to become distinguishable from the other parenchymatous cells and will form colourless cells in J. ascendens (Fig. 5A–C) and fusoid cells in the members of Poaceae that produce them (e.g. Figs 1E, F and 2A, B). This occurs in the same mesophyll region that differentiates the peg cells in F. indica (Fig. 6I, mature leaf) and irregular-shaped cells in Poaceae members with no development of fusoid cells (Fig. 6E–H, mature leaf). Stage 3: Final differentiation of tissues and the ongoing development of fusoid cells (LM and TEM) In J. ascendens, a single colourless cell (initial) divides to form three or four derivative cells (Fig. 5A–C); they then gradually increase in size along with the differentiation of the leaf blade and it is possible to observe the complete formation of their cell walls with LM (Fig. 5A–E). Fusoid cell differentiation in members of Poaceae herein studied occurs in much the same way; however, with LM we often observe the increase in size of the single fusoid cell, as in R. pilosa (Fig. 2A), A. aristulata (Fig. 2B inset), O. rzedowskiorum (Fig. 2E, H) and P. latifolius (Fig. 2G), with some content but no apparent cell divisions or, in the same sample in some taxa, cell divisions within the initial fusoid cell are clearly visible with LM [Fig. 2A (R. pilosa), Fig. 2C–E, H (O. rzedowskiorum) and Fig. 2F, G, I (P. latifolius)]. With TEM, we are able to easily observe division of the initial fusoid cell to form derivative cells, the vacuoles increasing in size with initial formation of cell plates, as in Parodiolyra micrantha (Fig. 3A–D), as well as in J. ascendens (not shown). A number of amyloplasts with starch grains in the developing fusoid cells are also often observed (e.g. Fig. 3E–G). At this point in time, peg cells, irregular-shaped cells and arm cells are almost all completely differentiated, and the rosette cells (abaxially positioned) in members of Bambusoideae (Poaceae) studied here become topographically distinct from the arm cells (e.g. Fig. 2B). Except for the fusoid cells, mesophyll differentiation often occurs simultaneously, but there are temporal differences in reaching complete maturity depending on the developmental characteristics of each species (e.g. earlier or later differentiation of mesophyll cells in relation to fusoid cell enlargement). Fusoid cells of adjacent bundles are virtually always separated by one or maybe two rosette cells in Bambusoideae members. Along with leaf enlargement, there is degradation of the middle lamella between adjacent fusoid cells within the same proximo-distal row and some chlorenchyma cells, and the fusoid cells are stretched and flattened along their long axis (along the lateral axis towards both margins of the leaf blade), as in P. latifolius (Fig. 2F, I, J). Stage 4: Final development of fusoid cells and mature leaves (LM and CSEM) Figure 4A–X shows many LM images of mature leaf blades of Poaceae members with fusoid cells, organized according to the most recent phylogenetic classification by Soreng et al. (2017), and two CSEM images of the mature leaf blade of G. angustifolia (Bambusoideae, Poaceae) mature leaf blade (Fig. 4Y, Z). Peg cells in F. indica (Fig. 6I), irregular-shaped cells in S. spicata (Fig. 4A), P. latifolius (Fig. 4B), Pooideae members (e.g. Fig. 4I, G. striata) and Panicoideae members (e.g. Fig. 4J, R. pilosa; Fig. 6E, S. scabrifolia), rosette cells in Oryzoideae members (e.g. Figs. 4D, Luziola spruceana; 4E, Oryza latifolia) and Bambusoideae members (e.g. Fig. 4F, P. micrantha; Fig. 4G, A. aristulata; Fig. 4H, O. rzedowskiorum), arm cells in Bambusoideae members (e.g. Fig. 4F, P. micrantha; 4G, A. aristulata; 4H, O. rzedowskiorum) and the colourless cells between vascular bundles (usually three or four) in J. ascendens (Fig. 5D) are completely differentiated (note that to better illustrate mature mesophyll cells, we included complementary images from species for which a developmental study was not herein performed). Yet, along with the breakdown of the middle lamella during leaf enlargement, fusoid cells (including any derivatives that may be present) gradually collapse along their long axis, leaving a cavity between adjacent successive fusoid cells (e.g. Fig. 4K–X, asterisks). In mature leaf blades, the fusoid cell in cross-section is a usually a cigar-shaped cavity (e.g. Fig. 4A–J, asterisks), and, due to the collapse, forms an I-shaped cell in longitudinal and paradermal views (e.g. Fig. 4K–Z, arrows). However, longitudinal and paradermal sections reveal that not all fusoid cells collapse (e.g. Fig. 4M–O, Q, R, T, W), since their cell walls still have a contact zone with the bundle sheath and other parenchymatous mesophyll cells (Fig. 4K–X, Supplementary Data Video). Also, as seen in paradermal view, fusoid cells comprising one longitudinal row of cells do not touch fusoid cells from any other row; however, they touch chlorenchyma cells and vascular sheath cells, and may touch adjacent fusoid cells within the same row if not collapsed (e.g. Fig. 4T–W, Supplementary Data Video) (note that ‘row’ refers to a set of many fusoid cells adjacent to each other along the entire lamina). DISCUSSION Fusoid cell origin and development Mesophyll with large colourless cells has been reported in Joinvilleaceae (Bayer and Appel, 1998), a family that currently comprises only two known species (J. ascendens and Joinvillea elegans). In this family, it is curious that colourless cells occur in the same mesophyll region as fusoid cells in the Poaceae, adjacent to and between vascular bundles. Also, we herein observed that the origin of a fusoid cell is from the ground meristem and thus is the same as a colourless cell in J. ascendens, even though there is developmental variation worth taking into account. In J. ascendens, a single colourless cell (initial) divides to form its derivatives and, as leaf maturation proceeds, initial and derivative cells do not collapse, remaining intact until their complete development. In the Poaceae species herein studied, however, the development of a fusoid cell occurs mostly in the same way as a colourless cell, but the initial and its derivative cells (internal divisions from a single fusoid cell with cell plates only) often collapse perpendicularly to the proximo-distal axis of the leaf blade later in their development, leaving cigar-shaped cavities in the mesophyll as seen in cross-section. Hence, the development of colourless and fusoid cells is quite similar and, considering their same topography and ground meristem origin, they are clearly homologous, as well as being homologous to the mesophyll cells adjacent to the vascular bundles in those species with no colourless or fusoid cells. Also, of note, colourless cells are more identifiable only when the vascular bundles are well differentiated. It is noteworthy that our results show that the origin of a fusoid cell is always from the ground meristem and that fusoid cells occur in seven subfamilies of the Poaceae, ubiquitously in the early-diverging lineages (Anomochlooideae, Pharoideae and Puelioideae), within the BOP clade (Oryzoideae, virtually all Bambusoideae, and Pooideae) and sporadically in the Panicoideae (PACMAD clade). Within the latter subfamily, the meristematic origin of the fusoid cells in Rugoloa (previously placed in Panicum) was thought to be distinct from the origin of fusoid cells in the early-diverging lineages and the BOP clade (Grass Phylogeny Working Group, 2001—the BOP clade referred to as the BEP clade in this work). In that work, these cells were assumed to be derived from laterally extended bundle sheaths and thus were referred to as fusoid-like cells. However, the homologies herein observed demonstrate that there is no reason to apply distinctive terminology for these cells in the Panicoideae. According to Sugimoto et al. (2011), transdifferentiation encompasses the process by which cells transform into another cell type outside of their already established differentiation paths. In contrast, a putative fusoid cell is clearly distinguishable from any other mesophyll cell even at the earliest stages and throughout development until its complete differentiation. The most recent work on the development of fusoid cells (in two species of Guadua) reported evidence of transdifferentiation of chlorenchyma cells into fusoid cells (Vega et al., 2016); however, the developmental work herein performed shows no evidence of transdifferentiation for the 13 species we sampled across the grass family. Vega et al. (2016) also described a conspicuous cell wall invagination during the differentiation of chlorenchyma cells into fusoid cells, which likewise was not herein observed. Since the cell wall invagination reported in that work is also evidenced in bundle sheath cells (see Fig. 1B, C in Vega et al., 2016), which do not have any lobes at maturity, it is very likely that the observed invaginations are a technical artefact caused by fixative methods. Of note, we had similar results in materials pre-fixed for <48 h. What appears to be a mature fusoid cell in leaf-blade cross-sections is herein interpreted as a cavity delimited by successive collapsed fusoid cells arranged perpendicularly to the veins (i.e. perpendicular to the proximo-distal axis in the lateral plane), with the caveat that sometimes fusoid cells do not collapse. A similar interpretation was given by Vega et al. (2016), referring to these apparent ‘cells’ as intercellular gas spaces delimited by successive collapsed fusoid cells. We consider ‘cavity’ to be the appropriate term because many Poaceae species have intercellular gas spaces in the mesophyll (not necessarily only as seen in cross-section and not in this configuration; e.g. Dengler et al., 1994; Gielwanowska et al., 2005), and thus it distinguishes more clearly between the types of intercellular spaces in order to avoid possible misinterpretation. Since fusoid cells collapse perpendicularly to their long axis but keep themselves partially connected to mesophyll cells adjacent to the epidermis and vascular sheath cells, they typically form cavities in the mesophyll rather than a complex network of small intercellular gas spaces. We propose that a single cavity as seen in a cross-section of a mature leaf blade is typically the result of the development and collapse of several fusoid cells (the initial and its internal derivative cells). During leaf development, we are not often able to perceive fusoid cell division with LM, and thus each of the successive I-shaped collapsed fusoid cells delimiting the cavities has always been interpreted as resulting from the collapse of a single fusoid cell. This is because the cell wall does not reach its full formation between derivative fusoid cells, often forming only cell plates (early stage), which is really difficult to observe with LM. Light microscopy techniques were able to clearly show internal fusoid cell divisions just in S. spicata (Anomochlooideae) and P. latifolius (Pharoideae); however, the division of a fusoid cell initial and its derivatives was observed with TEM techniques in all species herein studied that exhibit fusoid cells. Nonetheless, we cannot exclude the possibility that some initial fusoid cells simply enlarge and never divide internally before they collapse. In contrast, adjacent to the midrib, full formation of the cell walls of derivative cells does occur, albeit rarely, and thus several fusoid cells may be observed in this position in cross-section, as reported in mature leaves of Fargesia yunnanensis and Dendrocalamus giganteus (Wang et al., 2016). As noted by Ellis (1976, p. 105), due to the collapse of fusoid cells, longitudinal and paradermal sections of the leaf blade show these cells to have very narrow lumina in cross-section, having themselves collapsed in such a way that the tissue resembles a row of many ‘I’s’, which gives a lacunar aspect to the mesophyll (Jacques-Félix, 1962). We follow Ellis (1976) in describing these collapsed cells as I-shaped, even though they exhibit variation and can even appear dumbbell-shaped in some cases (e.g. Fig. 4W). As a final remark on development, the gradual collapse of fusoid cells observed in all studied species strongly suggests that they exhibit programmed cell death (PCD), as previously suggested for some Bambusoideae species (Vega et al., 2016; Wang et al., 2016). Quite often there is no evidence of a nucleus even at earlier stages of development as seen with LM, which implies an earlier PCD process, as previously suggested for F. yunnanensis (Wang et al., 2016). We will discuss PCD in more detail in a subsequent section. Phylogenetic implications of fusoid cells for the graminid clade The homology herein observed between the colourless cells in Joinvilleaceae and the fusoid cells in Poaceae is consistent with the relationships within the graminid clade as derived from molecular data (Givnish et al., 2010; Bouchenak-Khelladi et al., 2014). Within Flagellariaceae, which is sister to the rest of the graminid clade, there is no evidence of differentiated colourless or fusoid cells in the mesophyll. The Ecdeiocoleaceae do not produce developed leaves (Linder et al., 1998; Briggs, 2011), but regardless of whether it is sister to Joinvilleaceae or to Poaceae, the most parsimonious explanation is that the cell type ancestral to both colourless and fusoid cells evolved along the stem of the [(Joinvilleaceae + Ecdeiocoleaceae) + Poaceae] or [Joinvilleaceae + (Ecdeiocoleaceae + Poaceae)] clade. Further anatomical, developmental and molecular studies are needed to better document the origin of the fusoid cells in the graminid clade. The first attempt to produce a phylogenetic hypothesis of relationships within Poaceae was based exclusively on morphological data, in which Bambusoideae (including herbaceous tribes such as Anomochloeae, Phareae and Streptochaeteae) was interpreted as monophyletic based on the presence of arm cells and fusoid cells (Kellogg and Campbell, 1987). Clark et al. (1995), based on a family-wide analysis of plastid ndhF sequence data, resolved the three tribes mentioned above as forming two of the three early-diverging lineages within the family, with a more narrowly circumscribed Bambusoideae placed in the BOP clade. They therefore concluded that the presence of fusoid cells, a character previously useful for delimiting Bambusoideae, was probably a synapomorphy for Poaceae. A broader phylogenetic analysis of the Poaceae, incorporating both molecular sequence and morphological data, was elaborated by the Grass Phylogeny Working Group (2001) and clearly showed the fusoid cells as plesiomorphic within the family, but also typically found in Bambusoideae species and Streptogyna, then of uncertain placement in the BOP clade. Our results confirm the plesiomorphic presence of fusoid cells within Poaceae. As previously mentioned, fusoid cells occur in all three early-diverging lineages (Anomochlooideae, Pharoideae and Puelioideae), unevenly within the BOP clade (almost all Bambusoideae, but only in some Oryzoideae, and Pooideae), and in a few Panicoideae species. In the BOP clade, Oryzoideae is sister to Bambusoideae + Pooideae, and within Oryzoideae the tribe Streptogyneae is sister to the remainder of the subfamily: Ehrharteae, Oryzeae (including the subtribes Oryzinae and Zizaniinae) and Phyllorachideae (Soreng et al., 2015, 2017). Within Oryzoideae, fusoid cells are known only in the early-diverging Streptogyna (Streptogyneae) (Soderstrom et al., 1987) and in some species within the tribe Oryzeae, subtribe Zizaniinae (Tateoka, 1963; Kellogg, 2015; Leandro et al., 2016a) and subtribe Oryzinae (Oryza, this study). Within Pooideae, fusoid cells are reported only in Brachyelytrum (Barkworth et al., 2007; Stephenson and Saarela, 2007), both works, however, only mentioning (not accompanied by anatomical images) the presence of fusoid cells in the monotypic Brachyelytreae (herein not observed in B. erectum but documented in G. striata). Since the lack of fusoid cells in Brachyelytrum has also been reported in the literature (e.g. Tateoka 1957 – accompanied by a drawing of the leaf blade in cross-section; Campbell et al., 1986; Watson et al., 1992 onwards; Kellogg, 2015 – these latter three works also only mentioning the lack), the occurrence of fusoid cells in this genus is surely controversial and needs further investigation. In addition, for Glyceria, the occurrence of fusoid cells could be supported by the record of the sporadic presence of large cavities in the mesophyll of some species (Watson et al., 1992 onwards), which probably was interpreted in the same way as the mesophyll cavities observed, for instance, in the former Panicum sect. Laxa (Killeen and Clark, 1986), herein confirmed as being fusoid cells in R. pilosa (formerly Panicum pilosum). Hence, the presence of the fusoid cells in the early-diverging lineages, Streptogyneae, Zizaniinae, Oryziinae, a few Pooideae and generally in the Bambusoideae, and their lack in Ehrharteae, Phyllorachideae and most Pooideae clearly suggest multiple losses within the BOP clade (Kellogg, 2015). The same developmental origin of the fusoid cells herein confirmed between Panicoideae species and other Poaceae members is also relevant to phylogenetic inferences within the family. Fusoid cells have been reported in a few species of Rugoloa, Dallwatsonia (Killeen and Clark, 1986; Zuloaga et al., 1992 – both genera referred to as Panicum in this work), Homolepis (Watson et al., 1992 onwards) and Canastra (Morrone et al., 2001). These genera are all classified within the tribe Paspaleae, which, along with Paniceae and several other tribes with no fusoid cells reported, comprise the subfamily Panicoideae (Soreng et al., 2015, 2017). Thus, fusoid cells were probably lost early in the evolution of the PACMAD clade but regained in the Paspaleae. A broader sampling within Panicoideae as well as a formal character optimization of the presence of fusoid cells using the most recent molecular phylogeny of the family are needed to verify the evolution of this character within Poaceae. Some evolutionary and functional insights on fusoid cells based on TEM data Several possible functional roles for fusoid cells in mature leaves have been proposed, and can be summarized as structural (Vega et al., 2016), light scattering (March and Clark, 2011), photorespiration-related (Clark, 1991; Wang et al., 2016) and water dynamics-related (Vieira et al., 2002; Wang et al., 2016). These functions may not necessarily be mutually exclusive and thus may occur simultaneously in mature leaves. Hence, in this section we informally correlate these findings with environmental characteristics and our developmental results from young leaf blades. Estimates of divergence times for lineages within Poales vary, but there is agreement that Poales initially evolved mainly in open habitats and that the earliest transitions to shade habitats occurred in the graminid clade (Givnish et al., 2010; Bouchenak-Khelladi et al., 2014). The early-diverging lineages of Poaceae comprise plants growing in shaded forests or along forest margins, and more recent phylogenetic analyses based on plastomes suggest that the origin and early diversification of the BOP clade were associated with forested habitats as well (Grass Phylogeny Working Group II, 2012; Burke et al., 2016a, b). The presence of fusoid cells in the early-diverging grass lineages plus Streptogyneae and Bambusoideae suggests the correlation of these cells with shaded environments, although some forest grass lineages lack fusoid cells (e.g. Phyllorachideae of the Oryzoideae, and Diarrhena and apparently Brachyelytrum of the Pooideae in the BOP clade). In contrast, the presence of fusoid cells in some other Oryzoideae, comprising plants growing in open and often wet environments, suggests the influence of other environmental factors. The pattern with regard to habitat and the origin and diversification of the PACMAD clade is more ambiguous, even if the Panicoideae are inferred as the sister to the remainder of the clade (Burke et al., 2016b; Teisher et al., 2017). However, many early-diverging Panicoideae are associated with forest habitats but do not have fusoid cells (e.g. Centotheca, this study), although the facultative occurrence of fusoid cells has been reported in this genus (Watson et al., 1992 onwards, not accompanied by anatomical images), as well as lateral bundle sheath extensions in some centothecoid taxa (e.g. Soderstrom and Decker, 1973, also without any anatomical images), and at least some of the Paspaleae reported to have fusoid cells are found in open habitats (e.g. Canastra; Morrone et al., 2001). Since the facultative occurrence of fusoid cells has been reported in other grass species (e.g. Metcalfe, 1956; Wu, 1962; Pearson et al., 1994) and there is evidence of fusoid cells occurring in species associated with shaded and sunny habitats (e.g. March and Clark, 2011; Leandro et al., 2016a; Morrone et al., 2001), the occasional retention of fusoid cells in, for instance, Centotheca and Brachyelytrum would not be surprising but remains to be investigated. This background is important in understanding the potential roles played by the fusoid cells. Our developmental results show fusoid cells, which begin to enlarge and differentiate before the chorenchyma cells, with no chloroplasts but with several amyloplasts per fusoid cell, while the fusoid cells are still intact. Plants living in shaded environments often have wide leaves (Esau, 1977; Craine, 2009), presumably to maximize light absorption, which optimizes their survival under low levels of light. In this kind of environment, in general, there is suppression of photorespiration, with stomatal closure and consequent increase in plant growth (Walters and Reich, 1999; Craine, 2009). Thus, considering the low rates of photorespiration in shaded environments, along with our results showing no evidence of starch storage in the differentiating chlorenchyma cells, we argue that, at early stages of development, fusoid cells could act in the synthesis and storage of starch granules. In addition, the presence of plasmodesmata between chlorenchyma cells and fusoid cells, although few in number, supports the possibility of glucose transport from photosynthetic cells to fusoid cells, even though, according to Wang et al. (2016), no plasmodesmata or starch granules were associated with fusoid cells in F. yunnanensis. Of note, in this study, plasmodesmata in fusoid cells are observed only at earliest stages of mesophyll development. Further ultrastructural and functional studies should assist in the interpretation of the relationship of CO2 supply to the potential function of fusoid cells early in development. As previously mentioned, fusoid cell collapse (and apparent death) is often observed in mature leaves. At early stages of development, our TEM analysis reveals vacuoles increasing in size, internal division with initial formation of cell plates, and remains of membranes resulting from the lysis of organelles. Since most of these characteristics are typically described in plant cells undergoing PCD (Zhou et al., 2009), it is conceivable that the ultimate fate of these cells is determined by a PCD process, as previously suggested (Vega et al., 2016; Wang et al., 2016). Although our results support the presence of a PCD process during the development of fusoid cells, further investigation is needed to verify the occurrence of such a process. Conclusions During leaf differentiation, a single fusoid cell (initial) originating from the ground meristem may divide internally to form several fusoid cells (derivatives), although with formation of cell plates only. Our evidence suggests that this internal division occurs in many but not necessarily in all fusoid cells of a given leaf. In mature leaf blades as seen in cross-section, a fusoid cell is actually usually a cavity resulting from the collapse of the larger initial fusoid cell and its derivatives, most likely through a PCD process. Each cavity is delimited by successive collapsed fusoid cells arranged perpendicularly to the veins (i.e. perpendicular to the proximo-distal axis in the lateral plane) and along the entire lamina. Considering that they share the same topography and ground meristem origin, fusoid cells in Poaceae and colourless cells in Joinvilleaceae are homologous. Also, the meristematic origin of fusoid cells in Panicoideae is the same as in the early-diverging lineages and BOP clade (Poaceae), and thus they are homologous within the family. In addition, the presence of fusoid cells in the early-diverging lineages, Streptogyneae, Zizaniinae, a few Pooideae and Paspaleae and generally in the Bambusoideae, and their lack in the Ehrharteae, Phyllorachideae, Oryzinae and most Pooideae and Panicoideae, clearly suggest multiple losses within the BOP clade and an ancestral loss in the PACMAD clade with at least one regain in the Paspaleae (Panicoideae). We hypothesize that one role played by the fusoid cells is related to synthesis and storage of starch granules at early stages of leaf development. Further study is needed to verify functional analogies since this foundation is necessary to understand the evolution of mesophyll cells within the graminid clade. SUPPLEMENTARY DATA Supplementary data are available online at https://academic.oup.com/aob and consist of the following. S1: phylogenetic classification of Poaceae redrawn based on Soreng et al. (2015, 2017). S2: developmental stages of Aulonemia aristulata (Döll) McClure. Video: showing a tridimensional reconstruction of mature fusoid cells. ACKNOWLEDGEMENTS This research was completed as a partial fulfilment for the first author’s scholarship funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES (PDSE grant number 99999.003340/2015-05) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq (GD grant number 163550/2012-3; PDJ grant number 150797/2017-6 ). V.L.S. was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq (grant number 301692/2010-6). Field work, TEM and CSEM were supported by National Science Foundation grants DEB-1120750 and DEB-1342787 to L.G.C. The authors are grateful to Dr Harry T. Horner and Tracey Stewart for their assistance and also for access to the Roy J. Carver High Resolution Microscopy Facility (HRMF) at Iowa State University. Thanks to L.R.S. Tozin and the staff of the Centro de Microscopia Eletrônica (CME) at Universidade Estadual Paulista, IB, Botucatu, for their assistance with TEM techniques. LITERATURE CITED Angiosperm Phylogeny Group IV (APG). 2016. An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG IV. Botanical Journal of the Linnean Society  181: 1– 20. CrossRef Search ADS   Aliscioni SS, Giussani LM, Zuloaga FO, Kellogg EA. 2003. A molecular phylogeny of Panicum (Poaceae: Paniceae): tests of monophyly and phylogenetic placement within the Panicoideae. American Journal of Botany  90: 796– 821. Google Scholar CrossRef Search ADS   Aliscioni SS, Ospina JC, Gomiz NE. 2016. Morphology and leaf anatomy of Setaria s.l. (Poaceae: Panicoideae: Paniceae) and its taxonomic significance. 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Fusoid cells in the grass family Poaceae (Poales): a developmental study reveals homologies and suggests new insights into their functional role in young leaves

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Abstract

Abstract Background and Aims In mature grass leaf blades as seen in cross-section, oblong cell-like structures have been interpreted most recently as intercellular gas spaces delimited by successive collapsed fusoid cells. These cells have been reported in at least seven of 12 subfamilies of Poaceae and are considered a synapomorphy for the family; however, no developmental work has been performed to verify their meristematic origin or to assess possible homologies within the graminid clade (= Flagellariaceae + [(Joinvilleaceae + Ecdeiocoleaceae) + Poaceae]) or among subfamilies of Poaceae. A developmental study was therefore carried out, including 20 species in three families (Flagellariaceae, Joinvilleaceae and Poaceae), representing the earlier-diverging and derived branches within the graminid clade and Poaceae. Methods Light microscopy was combined with scanning electron microscopy, cryoscanning electron microscopy and transmission electron microscopy to study the development of leaves taken from the shoot apex of young plants. Mature leaf blades also were taken from living or dried plants and the mid-portion was studied. Key Results Developmental results show that, in mature leaf blades as seen in cross-section, one apparent fusoid cell is typically a cavity resulting from the collapse of the initial fusoid cell and its internal divisions, which are herein interpreted as derivative cells with formation of cell plates only. Each cavity is delimited by successive collapsed fusoid cells arranged perpendicularly to the veins. Fusoid cells in all studied Poaceae members originate from the ground meristem, as do the colourless cells in Joinvillea ascendens (Joinvilleaceae). These two types of mesophyll cell have a strongly similar ontogeny, distinguished mainly by the collapse of the fusoid cells in Poaceae, which is not observed in the colourless cells in J. ascendens. Conclusions Within the Poaceae, the meristematic origin of fusoid cells is the same in the early-diverging lineages, BOP clade and Panicoideae, and thus they are homologous within the family. The same topography and meristematic origin suggest that fusoid cells in Poaceae and colourless cells in Joinvilleaceae are homologous. The results also suggest that the role played by the fusoid cells in young grass leaves is related to synthesis and storage of starch granules at early stages of development. BOP clade, early-diverging grass lineages, Flagellariaceae, foliar anatomy, graminid clade, grasses, Joinvilleaceae, mesophyll cells, ontogeny, Panicoideae INTRODUCTION Poales is a diverse group that currently comprises 14 families with morphological, anatomical, embryological and molecular similarities (Stevens, 2001 onwards; Angiosperm Phylogeny Group IV, 2016). There are six clades within this order, of which the Flagellariaceae + [(Joinvilleaceae + Ecdeiocoleaceae) + Poaceae] is recognized as the graminid clade (Linder and Rudall, 2005); these four families share primarily morphological and embryological features but also are robustly supported as a clade based on plastome sequences (Stevens, 2001 onwards; Givnish et al., 2010). The graminid family Poaceae comprises nearly 12 000 species in 12 subfamilies widespread mainly in grasslands and forests all over the world (Grass Phylogeny Working Group II, 2012; Soreng et al., 2015, 2017), diversity for which anatomical studies have often provided useful features that aid in identification and delimitation at various taxonomic levels (e.g. Calderón and Soderstrom, 1973; Zuloaga et al., 1992, 1993; Aliscioni et al., 2003, 2016; Guglieri et al., 2008; Oliveira et al., 2008; Pelegrin et al., 2009; Leandro et al., 2016a), and also often indicate potential synapomorphies for clades (Judziewicz et al., 1999; Grass Phylogeny Working Group, 2001; Clark et al., 2015; Leandro et al., 2016b, 2017). Although knowledge of grass anatomy is constantly growing and there are many contributions in the literature, anatomical work on grasses has concentrated mainly on analyses of leaf-blade cross-sections using light microscopy. Strongly asymmetrically invaginated arm cells, intercostal fibres, Kranz anatomy and bundle spacing are examples of highly systematically informative leaf-blade features as seen in cross-section (e.g. Judziewicz et al., 1999; Grass Phylogeny Working Group, 2001; Viana et al., 2011; Grass Phylogeny Working Group II, 2012; Christin et al., 2013; Clark et al., 2015; Leandro et al., 2016b, 2017). In contrast, the systematic value of fusoid cells, an important leaf-blade anatomical feature for Poaceae, is still controversial in some instances and conflicting findings regarding their taxonomic distribution and functional role(s) in mature leaves have been published (see below). The term ‘fusoid cells’ was originally used by Metcalfe (1956), who studied the leaf-blade anatomy of many bamboo species (see the terminological history in Table 1). Even today, fusoid cells are assumed to be typical of Bambusoideae species, even though these cells are also observed in other subfamilies across the family (Grass Phylogeny Working Group, 2001): (1) the early-diverging lineages (Anomochlooideae, Pharoideae and Puelioideae) (Ellis, 1987—Anomochlooideae and Pharoideae referred to as Bambusoideae in this work; Clark et al., 2000); (2) the BOP clade (Bambusoideae and also in a few members of Oryzoideae and Pooideae) (Tateoka, 1963; Barkworth et al., 2007; Leandro et al., 2016a); and (3) the PACMAD clade (in a few members of Panicoideae) (Watson et al., 1985; Clayton and Renvoize, 1986; Killeen and Clark, 1986; Zuloaga et al., 1992). A summary phylogeny showing the relationships within the family and the occurrence of fusoid cells is provided as supplementary material (Supplementary Data S1). Table 1. Brief terminological history of fusoid cells Reference  Taxon  Anatomical term  Karelstschicoff (1868)  Dendrocalamus strictus  Faltenzellen, ‘folded cells’ (described as many thin-walled cells with different planes overarched)  Brandis (1907)  Bambusoideae  Apparent cavities (although described as cavities, they are recognized as cells)  Arber (1934)  Sasa disticha  Colourless central mesophyll cells  Page (1947)  Streptochaeta spicata  Enlarged mesophyll cells (with regard to the big cells in the mesophyll as seen in cross-section, longitudinal section and paradermal section)  Jacques-Félix (1954)  Bambusoideae  Lacune ou parenchyme différencié (with regard to the anastomosing network of canals that branch and then reunite as before)  Metcalfe (1956)  Bambusoideae  Fusoid cells (to define the fusiform-shaped cells as seen in cross-section)  Metcalfe (1960)  Bambusoideae  Fusoid cells  Wu (1960)  Bambuseae  Translucent fusoid cells (according to Metcalfe (1956), who defined these as fusoid cells)  Jacques-Félix (1962)  Oxytenanthera abyssinica  Cellules claires, ‘clear cells’  Renvoize (1985)  Bambusoideae  Fusoid cells  Killeen and Clark (1986)  Panicum sect. Laxa  Fusoid-like cells similar to cavities that apparently are derived from laterally extended bundle sheaths  Renvoize (1987)  Bambuseae  fusoid cells  Clark (1991)  Bambusoideae  Fusoid cells or spaces formed by their collapse (with regard to the spaces as seen in cross-section and longitudinal section)  Zuloaga et al. (1992)  Panicum subg. Phanophyrum sect. Laxa  Translucent fusoid cells, fusoid cells, and fusoid cavities (used in the same work to refer to the same cell type)  Grass Phylogeny Working Group (2001)  Poaceae  Fusoid cells for early-diverging lineages and Bambusoideae, and fusoid-like cells for Panicoideae  Vega et al. (2016)  Guadua species  Intercellular gas spaces (with regard to fusoid cell collapse and the space left between adjacent fusoid cells)  Wang et al. (2016)  Bambusoideae  Fusoid cells  Reference  Taxon  Anatomical term  Karelstschicoff (1868)  Dendrocalamus strictus  Faltenzellen, ‘folded cells’ (described as many thin-walled cells with different planes overarched)  Brandis (1907)  Bambusoideae  Apparent cavities (although described as cavities, they are recognized as cells)  Arber (1934)  Sasa disticha  Colourless central mesophyll cells  Page (1947)  Streptochaeta spicata  Enlarged mesophyll cells (with regard to the big cells in the mesophyll as seen in cross-section, longitudinal section and paradermal section)  Jacques-Félix (1954)  Bambusoideae  Lacune ou parenchyme différencié (with regard to the anastomosing network of canals that branch and then reunite as before)  Metcalfe (1956)  Bambusoideae  Fusoid cells (to define the fusiform-shaped cells as seen in cross-section)  Metcalfe (1960)  Bambusoideae  Fusoid cells  Wu (1960)  Bambuseae  Translucent fusoid cells (according to Metcalfe (1956), who defined these as fusoid cells)  Jacques-Félix (1962)  Oxytenanthera abyssinica  Cellules claires, ‘clear cells’  Renvoize (1985)  Bambusoideae  Fusoid cells  Killeen and Clark (1986)  Panicum sect. Laxa  Fusoid-like cells similar to cavities that apparently are derived from laterally extended bundle sheaths  Renvoize (1987)  Bambuseae  fusoid cells  Clark (1991)  Bambusoideae  Fusoid cells or spaces formed by their collapse (with regard to the spaces as seen in cross-section and longitudinal section)  Zuloaga et al. (1992)  Panicum subg. Phanophyrum sect. Laxa  Translucent fusoid cells, fusoid cells, and fusoid cavities (used in the same work to refer to the same cell type)  Grass Phylogeny Working Group (2001)  Poaceae  Fusoid cells for early-diverging lineages and Bambusoideae, and fusoid-like cells for Panicoideae  Vega et al. (2016)  Guadua species  Intercellular gas spaces (with regard to fusoid cell collapse and the space left between adjacent fusoid cells)  Wang et al. (2016)  Bambusoideae  Fusoid cells  View Large Table 1. Brief terminological history of fusoid cells Reference  Taxon  Anatomical term  Karelstschicoff (1868)  Dendrocalamus strictus  Faltenzellen, ‘folded cells’ (described as many thin-walled cells with different planes overarched)  Brandis (1907)  Bambusoideae  Apparent cavities (although described as cavities, they are recognized as cells)  Arber (1934)  Sasa disticha  Colourless central mesophyll cells  Page (1947)  Streptochaeta spicata  Enlarged mesophyll cells (with regard to the big cells in the mesophyll as seen in cross-section, longitudinal section and paradermal section)  Jacques-Félix (1954)  Bambusoideae  Lacune ou parenchyme différencié (with regard to the anastomosing network of canals that branch and then reunite as before)  Metcalfe (1956)  Bambusoideae  Fusoid cells (to define the fusiform-shaped cells as seen in cross-section)  Metcalfe (1960)  Bambusoideae  Fusoid cells  Wu (1960)  Bambuseae  Translucent fusoid cells (according to Metcalfe (1956), who defined these as fusoid cells)  Jacques-Félix (1962)  Oxytenanthera abyssinica  Cellules claires, ‘clear cells’  Renvoize (1985)  Bambusoideae  Fusoid cells  Killeen and Clark (1986)  Panicum sect. Laxa  Fusoid-like cells similar to cavities that apparently are derived from laterally extended bundle sheaths  Renvoize (1987)  Bambuseae  fusoid cells  Clark (1991)  Bambusoideae  Fusoid cells or spaces formed by their collapse (with regard to the spaces as seen in cross-section and longitudinal section)  Zuloaga et al. (1992)  Panicum subg. Phanophyrum sect. Laxa  Translucent fusoid cells, fusoid cells, and fusoid cavities (used in the same work to refer to the same cell type)  Grass Phylogeny Working Group (2001)  Poaceae  Fusoid cells for early-diverging lineages and Bambusoideae, and fusoid-like cells for Panicoideae  Vega et al. (2016)  Guadua species  Intercellular gas spaces (with regard to fusoid cell collapse and the space left between adjacent fusoid cells)  Wang et al. (2016)  Bambusoideae  Fusoid cells  Reference  Taxon  Anatomical term  Karelstschicoff (1868)  Dendrocalamus strictus  Faltenzellen, ‘folded cells’ (described as many thin-walled cells with different planes overarched)  Brandis (1907)  Bambusoideae  Apparent cavities (although described as cavities, they are recognized as cells)  Arber (1934)  Sasa disticha  Colourless central mesophyll cells  Page (1947)  Streptochaeta spicata  Enlarged mesophyll cells (with regard to the big cells in the mesophyll as seen in cross-section, longitudinal section and paradermal section)  Jacques-Félix (1954)  Bambusoideae  Lacune ou parenchyme différencié (with regard to the anastomosing network of canals that branch and then reunite as before)  Metcalfe (1956)  Bambusoideae  Fusoid cells (to define the fusiform-shaped cells as seen in cross-section)  Metcalfe (1960)  Bambusoideae  Fusoid cells  Wu (1960)  Bambuseae  Translucent fusoid cells (according to Metcalfe (1956), who defined these as fusoid cells)  Jacques-Félix (1962)  Oxytenanthera abyssinica  Cellules claires, ‘clear cells’  Renvoize (1985)  Bambusoideae  Fusoid cells  Killeen and Clark (1986)  Panicum sect. Laxa  Fusoid-like cells similar to cavities that apparently are derived from laterally extended bundle sheaths  Renvoize (1987)  Bambuseae  fusoid cells  Clark (1991)  Bambusoideae  Fusoid cells or spaces formed by their collapse (with regard to the spaces as seen in cross-section and longitudinal section)  Zuloaga et al. (1992)  Panicum subg. Phanophyrum sect. Laxa  Translucent fusoid cells, fusoid cells, and fusoid cavities (used in the same work to refer to the same cell type)  Grass Phylogeny Working Group (2001)  Poaceae  Fusoid cells for early-diverging lineages and Bambusoideae, and fusoid-like cells for Panicoideae  Vega et al. (2016)  Guadua species  Intercellular gas spaces (with regard to fusoid cell collapse and the space left between adjacent fusoid cells)  Wang et al. (2016)  Bambusoideae  Fusoid cells  View Large Although fusoid cells are recognized as a prominent feature of the early-diverging lineages and Bambusoideae, the role played by these cells in mature leaves has never been satisfactorily explained. Clark (1991) proposed that fusoid cells act as reservoirs for CO2 from photorespiration, a hypothesis later rejected by March and Clark (2011) based on the typically lower rates of photorespiration in shaded environments and the greater development of fusoid cells in shade leaves. Rather, March and Clark (2011) proposed that cavities formed by the collapse of fusoid cells may be related to intra- or intercellular reflectance, which contributes to the absorption and intrafoliar redistribution of light in shaded environments. Vega et al. (2016) proposed that the I-shaped collapsed fusoid cells as seen in longitudinal section may play a structural role, supporting the mature leaf-blade architecture. Vieira et al. (2002) and Wang et al. (2016), in turn, suggested the relationship of fusoid cells to storage, transportation and water balance functions. Wang et al. (2016) also reinforced the potential role of fusoid cells as CO2 reservoirs from photorespiration based on greenhouse experiments under high temperatures. Given their wide occurrence across Poaceae, fusoid cells have been considered an important character in taxonomic descriptions and also in establishing phylogenetic relationships within the family. On the other hand, previous work has shown that these cells are environmentally influenced and thus their occurrence may be facultative within the same species or even within the same sample or individual (e.g. Metcalfe, 1956; Wu, 1962; Pearson et al., 1994; March and Clark, 2011). For example, in bamboos, plants living in sunny habitats often lack these cells, whereas the same species growing in shade always have them (e.g. March and Clark, 2011); in contrast, Oryzeae (Oryzoideae) comprise aquatic or mesic species, which may exhibit fusoid cells, but primarily live in open habitats (Tateoka, 1963; Leandro et al., 2016a). In mature leaf blades as seen in cross-section, the fusoid cells are large, mainly more or less oblong with thin cell walls, and apparently with no content (Grass Phylogeny Working Group, 2001; Kellogg, 2015). This set of features makes these cells visually similar to cavities or air spaces in the mesophyll, consequently generating doubts about whether they are cells or intercellular spaces (Karelstschicoff, 1868; Brandis, 1907). Their cellular nature was confirmed in studies of leaf-blade development in Streptochaeta (Page, 1947) and the bamboo Oxytenanthera abyssinica (Jacques-Félix, 1962), in which the fusoid cells were called ‘enlarged mesophyll cells’ and ‘cellules claires’, respectively (Table 1). A recent study of the development of fusoid cells in two species of Guadua (Bambusoideae) show that these cells differentiate, enlarge and then usually collapse to form intercellular gas spaces as seen in cross-section (Vega et al., 2016; Table 1). These three developmental studies, although addressing aspects of fusoid cell ontogeny, do not completely clarify the origin of these cells or their occurrence or homology across the grass family. Since there are no leaf developmental studies addressing the occurrence of fusoid cells and their homologies among families within the graminid clade, there is no consensus with regard to a number of questions: (1) whether the presence of fusoid cells is in fact a synapomorphy for the Poaceae (Grass Phylogeny Working Group, 2001), considering their occurrence in the early-diverging lineages and the presence of colourless cells similar to fusoid cells in the mesophyll of Joinvilleaceae members; (2) whether fusoid cells were lost multiple times within the BOP clade, considering the absence of fusoid cells in many Oryzoideae and in most Pooideae members (Grass Phylogeny Working Group, 2001); and (3) whether their origin is the same across the entire family – they are called ‘fusoid-like cells’ in the Panicoideae (Killeen and Clark, 1986). Hence, given the importance of fusoid cells for grass systematics and phylogeny, the primary aims of this work were: (1) to study leaf-blade development within Poaceae focusing on the development of fusoid cells in order to verify their meristematic origin and putative homologies; and (2) to determine whether the colourless cells of the mesophyll similar to fusoid cells observed in Joinvilleaceae have the same origin as in the Poaceae. To that end, we studied the development of 20 species among Poaceae, Joinvilleaceae and Flagellariaceae (the graminid clade). MATERIALS AND METHODS Sampling For the developmental work the following taxa were studied: (1) Joinvillea ascendens (Joinvilleaceae); and (2) several Poaceae members known to have fusoid cells in six subfamilies: the two earliest-diverging lineages (Anomochlooideae and Pharoideae), the BOP clade (Bambusoideae, Oryzoideae and Pooideae) and the PACMAD clade (Panicoideae). Since we examined leaf-blade development in Joinvilleaceae in order to compare it with Poaceae, we also included Flagellaria indica (Flagellariaceae), placed within the graminid clade and sister to [(Joinvilleaceae + Ecdeiocoleaceae) + Poaceae] (Stevens, 2001 onwards; Linder and Rudall, 2005). To define homologies properly we also included selected species across the grass family in which fusoid cells are lacking. Living plants were sampled in their natural habitat or from the R. W. Pohl Conservatory at Iowa State University, USA. We were not able to collect living plants to represent the subfamily Puelioideae (from Tropical Africa), but we included anatomical results from mature dried leaves of Guaduella oblonga (Puelioideae) and also from other Poaceae species in order to provide a broad perspective. All species included in this work, sampling method, purpose of study and vouchers are provided in Table 2. Note that although there are differences in mesophyll arrangement and vascular bundle spacing between C3 and C4 species (Dengler et al., 1994; Ueno et al., 2006), Setaria scabrifolia (C4) was considered in this study since our main aim was only to compare the development of ground meristem cells into types of mesophyll cells, especially fusoid cells. Table 2. List of studied species, occurrence of fusoid cells, vouchers and types of microscopy and analysis. Taxon  Fusoid cells  Voucher  Analysis performed        LM  EM        DV  AN  CS  T  Flagellariaceae   Flagellaria indica L.  –  Clark & Zhang 1305 (ISC)  X  X      Joinvilleaceae   Joinvillea ascendens Gaudich. ex Brongn. & Gris  –  Clark & Attigala 1714 (ISC)  X  X    X  Poaceae   Anomochlooideae    Streptochaeta spicata Schrad. ex Nees  +  Clark & Lewis 1642 (ISC)  X  X    X   Pharoideae    Pharus latifolius L.  +  Klahs 1250 (ISC)  X  X    X   Puelioideae    Guaduella oblonga Hutchinson ex W. D. Clayton  +  Baldwin 6704 (ISC)    X    X   Oryzoideae    Ehrharta erecta Lam.  –  Clark & Gallaher (ISC)  X  X        Luziola spruceana Benth. ex Döll  +  Moreira 249 (CGMS)    X        Oryza latifolia Desv.  +  Guglieri-Caporal 3172 (CGMS)    X       Bambusoideae    Raddia brasiliensis Bertol.  +  Clark & Attigala 1713 (ISC)  X  X        Parodiolyra micrantha (Kunth) Davidse & Zuloaga  +  Shirasuna 2863 (SP)    X        Aulonemia aristulata (Döll) McClure  +  Shirasuna 2860 (SP)  X  X    X    Chusquea attenuata (Döll) L.G. Clark  +  Campos et al. s.n. (ISC)  X          Guadua angustifolia Kunth  +  Clark 1095 (ISC)      X      Otatea rzedowskiorum Ruiz-Sanchez  +  Clark s.n. (ISC)  X  X       Pooideae    Brachyelytrum erectum (Schreb.) P. Beauv.  –  Clark & Dixon 1669 (ISC)  X  X        Diarrhena obovata (Gleason) Brandenburg  –  Clark & Dixon 1667 (ISC)  X  X        Glyceria striata (Lam.) Hitchc.  +  Clark & Dixon 1671 (ISC)  X  X       Panicoideae    Centotheca lappacea (L.) Desv.  –  Sanchez-Ken s.n. (ISC)  X  X        Rugoloa pilosa (Sw.) Zuloaga  +  Leandro 161 (CGMS)  X  X        Setaria scabrifolia (Nees) Kunth  –  Leandro 160 (CGMS)  X  X      Taxon  Fusoid cells  Voucher  Analysis performed        LM  EM        DV  AN  CS  T  Flagellariaceae   Flagellaria indica L.  –  Clark & Zhang 1305 (ISC)  X  X      Joinvilleaceae   Joinvillea ascendens Gaudich. ex Brongn. & Gris  –  Clark & Attigala 1714 (ISC)  X  X    X  Poaceae   Anomochlooideae    Streptochaeta spicata Schrad. ex Nees  +  Clark & Lewis 1642 (ISC)  X  X    X   Pharoideae    Pharus latifolius L.  +  Klahs 1250 (ISC)  X  X    X   Puelioideae    Guaduella oblonga Hutchinson ex W. D. Clayton  +  Baldwin 6704 (ISC)    X    X   Oryzoideae    Ehrharta erecta Lam.  –  Clark & Gallaher (ISC)  X  X        Luziola spruceana Benth. ex Döll  +  Moreira 249 (CGMS)    X        Oryza latifolia Desv.  +  Guglieri-Caporal 3172 (CGMS)    X       Bambusoideae    Raddia brasiliensis Bertol.  +  Clark & Attigala 1713 (ISC)  X  X        Parodiolyra micrantha (Kunth) Davidse & Zuloaga  +  Shirasuna 2863 (SP)    X        Aulonemia aristulata (Döll) McClure  +  Shirasuna 2860 (SP)  X  X    X    Chusquea attenuata (Döll) L.G. Clark  +  Campos et al. s.n. (ISC)  X          Guadua angustifolia Kunth  +  Clark 1095 (ISC)      X      Otatea rzedowskiorum Ruiz-Sanchez  +  Clark s.n. (ISC)  X  X       Pooideae    Brachyelytrum erectum (Schreb.) P. Beauv.  –  Clark & Dixon 1669 (ISC)  X  X        Diarrhena obovata (Gleason) Brandenburg  –  Clark & Dixon 1667 (ISC)  X  X        Glyceria striata (Lam.) Hitchc.  +  Clark & Dixon 1671 (ISC)  X  X       Panicoideae    Centotheca lappacea (L.) Desv.  –  Sanchez-Ken s.n. (ISC)  X  X        Rugoloa pilosa (Sw.) Zuloaga  +  Leandro 161 (CGMS)  X  X        Setaria scabrifolia (Nees) Kunth  –  Leandro 160 (CGMS)  X  X      CGMS, Herbarium of Universidade Federal de Mato Grosso do Sul; ISC, Ada Hayden Herbarium of Iowa State University; SP, Herbarium of the Instituto de Botânica de São Paulo. AN, anatomical work (mature leaves); DV, developmental work (ontogeny); LM, light microscopy; EM, electron microscopy; CS, cryoscanning electron microscopy; T, transmission electron microscopy. +, presence; –, absence. View Large Table 2. List of studied species, occurrence of fusoid cells, vouchers and types of microscopy and analysis. Taxon  Fusoid cells  Voucher  Analysis performed        LM  EM        DV  AN  CS  T  Flagellariaceae   Flagellaria indica L.  –  Clark & Zhang 1305 (ISC)  X  X      Joinvilleaceae   Joinvillea ascendens Gaudich. ex Brongn. & Gris  –  Clark & Attigala 1714 (ISC)  X  X    X  Poaceae   Anomochlooideae    Streptochaeta spicata Schrad. ex Nees  +  Clark & Lewis 1642 (ISC)  X  X    X   Pharoideae    Pharus latifolius L.  +  Klahs 1250 (ISC)  X  X    X   Puelioideae    Guaduella oblonga Hutchinson ex W. D. Clayton  +  Baldwin 6704 (ISC)    X    X   Oryzoideae    Ehrharta erecta Lam.  –  Clark & Gallaher (ISC)  X  X        Luziola spruceana Benth. ex Döll  +  Moreira 249 (CGMS)    X        Oryza latifolia Desv.  +  Guglieri-Caporal 3172 (CGMS)    X       Bambusoideae    Raddia brasiliensis Bertol.  +  Clark & Attigala 1713 (ISC)  X  X        Parodiolyra micrantha (Kunth) Davidse & Zuloaga  +  Shirasuna 2863 (SP)    X        Aulonemia aristulata (Döll) McClure  +  Shirasuna 2860 (SP)  X  X    X    Chusquea attenuata (Döll) L.G. Clark  +  Campos et al. s.n. (ISC)  X          Guadua angustifolia Kunth  +  Clark 1095 (ISC)      X      Otatea rzedowskiorum Ruiz-Sanchez  +  Clark s.n. (ISC)  X  X       Pooideae    Brachyelytrum erectum (Schreb.) P. Beauv.  –  Clark & Dixon 1669 (ISC)  X  X        Diarrhena obovata (Gleason) Brandenburg  –  Clark & Dixon 1667 (ISC)  X  X        Glyceria striata (Lam.) Hitchc.  +  Clark & Dixon 1671 (ISC)  X  X       Panicoideae    Centotheca lappacea (L.) Desv.  –  Sanchez-Ken s.n. (ISC)  X  X        Rugoloa pilosa (Sw.) Zuloaga  +  Leandro 161 (CGMS)  X  X        Setaria scabrifolia (Nees) Kunth  –  Leandro 160 (CGMS)  X  X      Taxon  Fusoid cells  Voucher  Analysis performed        LM  EM        DV  AN  CS  T  Flagellariaceae   Flagellaria indica L.  –  Clark & Zhang 1305 (ISC)  X  X      Joinvilleaceae   Joinvillea ascendens Gaudich. ex Brongn. & Gris  –  Clark & Attigala 1714 (ISC)  X  X    X  Poaceae   Anomochlooideae    Streptochaeta spicata Schrad. ex Nees  +  Clark & Lewis 1642 (ISC)  X  X    X   Pharoideae    Pharus latifolius L.  +  Klahs 1250 (ISC)  X  X    X   Puelioideae    Guaduella oblonga Hutchinson ex W. D. Clayton  +  Baldwin 6704 (ISC)    X    X   Oryzoideae    Ehrharta erecta Lam.  –  Clark & Gallaher (ISC)  X  X        Luziola spruceana Benth. ex Döll  +  Moreira 249 (CGMS)    X        Oryza latifolia Desv.  +  Guglieri-Caporal 3172 (CGMS)    X       Bambusoideae    Raddia brasiliensis Bertol.  +  Clark & Attigala 1713 (ISC)  X  X        Parodiolyra micrantha (Kunth) Davidse & Zuloaga  +  Shirasuna 2863 (SP)    X        Aulonemia aristulata (Döll) McClure  +  Shirasuna 2860 (SP)  X  X    X    Chusquea attenuata (Döll) L.G. Clark  +  Campos et al. s.n. (ISC)  X          Guadua angustifolia Kunth  +  Clark 1095 (ISC)      X      Otatea rzedowskiorum Ruiz-Sanchez  +  Clark s.n. (ISC)  X  X       Pooideae    Brachyelytrum erectum (Schreb.) P. Beauv.  –  Clark & Dixon 1669 (ISC)  X  X        Diarrhena obovata (Gleason) Brandenburg  –  Clark & Dixon 1667 (ISC)  X  X        Glyceria striata (Lam.) Hitchc.  +  Clark & Dixon 1671 (ISC)  X  X       Panicoideae    Centotheca lappacea (L.) Desv.  –  Sanchez-Ken s.n. (ISC)  X  X        Rugoloa pilosa (Sw.) Zuloaga  +  Leandro 161 (CGMS)  X  X        Setaria scabrifolia (Nees) Kunth  –  Leandro 160 (CGMS)  X  X      CGMS, Herbarium of Universidade Federal de Mato Grosso do Sul; ISC, Ada Hayden Herbarium of Iowa State University; SP, Herbarium of the Instituto de Botânica de São Paulo. AN, anatomical work (mature leaves); DV, developmental work (ontogeny); LM, light microscopy; EM, electron microscopy; CS, cryoscanning electron microscopy; T, transmission electron microscopy. +, presence; –, absence. View Large Developmental study (young leaves) Light microscopy (LM). Leaves were taken from the shoot apex of young plants, fixed in FAA50 (formaldehyde–acetic acid–ethanol) for 48 h (Johansen, 1940) and then stored in 70 % ethanol. Each sample was dehydrated through a graded n-butyl series and embedded in glycol methacrylate (Leica Historesin Embedding Kit) following conventional methods. Cross-sections were obtained with a rotary microtome (Leica RM 2145 or Spencer 820) and stained with toluidine blue (Feder and O’Brien, 1968) to visualize cellulosic cell walls plus periodic acid–Schiff (PAS) to provide better contrast. Permanent slides were mounted with Entellan (Merck, Germany) or Permount (Fisher, USA). Transmission electron microscopy (TEM). Small leaf-blade pieces (1 mm diameter) from the shoot apex of young plants were fixed with 2 % paraformaldehyde/2 % glutaraldehyde in a 0.1 m cacodylate buffer at 4 ºC for 48 h (Horner, 2012). The pieces were buffer-washed three times for a total of 1 h, dehydrated through an ethanol series, and embedded in Spurr’s resin (Spurr Low-Viscosity Embedding Kit). Ultra-thin sections were cut with a diamond knife then placed on copper grids and post-stained with lead and uranium. Anatomical study (mature leaf blades) Light microscopy. Mature leaf blades were taken from living or dried plants and sampled from the middle portion of the blade. For living plants, each sample was fixed in FAA50 for 48 h (Johansen, 1940) and stored in 70 % ethanol. Due to the amount of sclerified and silicified tissues, sections were made by using two distinct methods: (1) leaf-blade pieces (0.5 cm2) were embedded in polyethylene glycol 1500 solution and kept in an incubator at 60 ºC for 15 d, dried and then sectioned (adapted from Richter, 1985); or (2) samples were treated with 30 % hydrofluoric acid for 72 h, dehydrated through a graded n-ethanol series, embedded in paraffin and then sectioned. For both methods, sections were made using a Leica RM 2145 or Spencer 820 rotary microtome and then stained with epoxy tissue stain (Spurlock et al., 1966). Permanent slides were mounted with Entellan (Merck, Germany). Also, pieces of ~0.5 cm2 from the middle portion of dried leaf blades of Chusquea attenuata were removed for analysis of fusoid cells through the depth of the leaf blade. These pieces were hydrated through a graded series of ethyl alcohol (50, 25 and 10 %) and then soaked in 1:1/dH20 (deionized water) until the samples were translucent. Samples were rinsed with dH20, dehydrated through a graded series of ethyl alcohol (25, 50 and 70 %) and then stained with safranin and fast green (Johansen, 1940). Stained samples were treated with ethyl alcohol (95 and 100 %), xylene and then xylene and Permount (1:1). Permanent slides were mounted with Permount (Fisher, USA). Cryoscanning electron microscopy (CSEM). Pieces of ~ 1 cm2 from the middle portion of living leaf blades of Guadua angustifolia were vertically attached to a cylindrical sample holder (stub) with cryoglue (Tissue-Tec, Scigen Scientific, USA) and then frozen in a bath of liquid nitrogen with vacuum applied. These samples were placed in a cryopreparation chamber (Quorum Cryo System PPT 3010-T) and then cryofractured with a semirotary cryoknife to expose the mesophyll tissues in cross-section. Description, observation, images and tridimensional reconstruction Descriptions and illustrations mainly concentrated on the development of fusoid cells and the terminology primarily followed Ellis (1976, 1979). In order to better present the results, the progression of leaf development was divided into four stages. For LM analyses, photomicrographs were obtained with a Zeiss Axio Observer microscope using ZEN 2.0 blue software or with a Leica DM4000B microscope using the Leica Application Suite LASV4.0. Scanning/transmission electron microscope images (STEM) were captured with a JEOL 2100 or FEI Tecnai™ Spirit TEM. In order to build a paradermal scan with tridimensional reconstruction showing the fusoid cells through the depth of the leaf blade, each 0.5-μm-thick section of a leaf-blade clearing of C. attenuata was photographed along the Z-axis to create a stack of eight to ten 0.5-μm-thick optical sections. Z-stacks from each section were aligned and modelled also using ZEN 2.0 blue. The analyses herein described were performed in the Departamento de Botânica at UNESP-Rio Claro, Brazil, in the Centro de Microscopia Eletrônica (CME) at UNESP-Botucatu, Brazil, and in the Roy J. Carver High Resolution Microscopy Facility (HRMF) at Iowa State University, USA. RESULTS The following descriptions include four stages of leaf development with an emphasis on the fusoid cells (Figs 1A–F, 2A–J, 3A–G and 4A–Z, Poaceae). Images for the development of leaves with colourless cells (Fig. 5A–E, Joinvilleaceae) and with no fusoid cells (Fig. 6A–I, Poaceae and Flagellariaceae) are included, as well as a video showing a tridimensional reconstruction of mature fusoid cells (Supplementary Data Video). The text and figures mix descriptions of several studied species, but a full developmental series of Aulonemia aristulata, a Bambusoideae member that exhibits a typical development of fusoid cells, is provided as supplementary material (Supplementary Data S2). Fig. 1. View largeDownload slide Cross-sections of leaf blades (LM) at early stages of development with emphasis on the development of fusoid cells in Poaceae species. (A, B, D, E) Aulonemia aristulata (Bambusoideae); (C, F) Rugoloa pilosa (Panicoideae). Red arrows show cell divisions, blue coloration highlights procambial strands (pr) and pink coloration highlights the differentiation of ground meristem (gm) into fusoid cells. (A) Early procambial strand of the midvein, protoderm (pt) and ground meristem. (B) Midvein and initial development of minor procambial strands. (C) Minor procambial strands are formed between pre-existing major ones. (D) Major and minor procambial strands. (E) Subsequent differentiation of procambial strands into vascular bundles and ground meristem cells into fusoid cells. (F) Differentiation of protoderm and procambium, as well as ground meristem cells with fusoid cells readily distinguishable. OBS: For additional information, see supplementary note provided in the Supplementary Data S2. Scale bars = 15 µm. Fig. 1. View largeDownload slide Cross-sections of leaf blades (LM) at early stages of development with emphasis on the development of fusoid cells in Poaceae species. (A, B, D, E) Aulonemia aristulata (Bambusoideae); (C, F) Rugoloa pilosa (Panicoideae). Red arrows show cell divisions, blue coloration highlights procambial strands (pr) and pink coloration highlights the differentiation of ground meristem (gm) into fusoid cells. (A) Early procambial strand of the midvein, protoderm (pt) and ground meristem. (B) Midvein and initial development of minor procambial strands. (C) Minor procambial strands are formed between pre-existing major ones. (D) Major and minor procambial strands. (E) Subsequent differentiation of procambial strands into vascular bundles and ground meristem cells into fusoid cells. (F) Differentiation of protoderm and procambium, as well as ground meristem cells with fusoid cells readily distinguishable. OBS: For additional information, see supplementary note provided in the Supplementary Data S2. Scale bars = 15 µm. Fig. 2. View largeDownload slide Cross-sections of leaf blades (LM) at early stages of development with emphasis on the development of fusoid cells in Poaceae species. (A) Rugoloa pilosa (Panicoideae); (B) Aulonemia aristulata (Bambusoideae); (C, D, E, H) Otatea rzedowskiorum (Bambusoideae); (F, G, I, J) Pharus latifolius (Pharoideae). Red arrows show cell divisions, pink coloration highlights the differentiation of ground meristem into fusoid cells, and asterisks show the initial cavities formed from the degradation of the middle lamella and gradual collapse of fusoid cells. (A) Adaxial (ad) and abaxial (ab) epidermis and vascular bundles (vb) differentiated (except the bulliform cells), and differentiation of ground meristem cells with fusoid cells readily distinguishable. (B) Several fusoid cells adjacent to the vascular bundles and inset showing a young fusoid cell with content. (C) Initial fusoid cell giving rise to a derivative fusoid cell (internal division within an initial fusoid cell). (D) Subsequent division from initial and derivative fusoid cells (internal divisions within an initial fusoid cell). (E) Vascular bundle between one (left) and three (right) fusoid cells, with nuclei visible in the fusoid cells. (F) Degradation of the middle lamella between fusoid and chlorenchyma cells. (G, H) Fusoid cell divisions accompanying vascular bundle maturation. Note the fully differentiated vascular tissue and enlarged bundle sheath cells in (G). (I) Degradation of the middle lamella between fusoid and chlorenchyma cells following bundle maturation. (J) Fusoid cell stretched and flattened along the lateral axis as a result of the degradation of the middle lamella and leaf blade enlargement. OBS: For additional information, see Supplementary Data S2. Scale bars (C, D, E, H) = 10 µm; (A) =15 µm; (I) = 30 µm; (F, G, J) = 40 µm; (B, inset) = 50 µm. Fig. 2. View largeDownload slide Cross-sections of leaf blades (LM) at early stages of development with emphasis on the development of fusoid cells in Poaceae species. (A) Rugoloa pilosa (Panicoideae); (B) Aulonemia aristulata (Bambusoideae); (C, D, E, H) Otatea rzedowskiorum (Bambusoideae); (F, G, I, J) Pharus latifolius (Pharoideae). Red arrows show cell divisions, pink coloration highlights the differentiation of ground meristem into fusoid cells, and asterisks show the initial cavities formed from the degradation of the middle lamella and gradual collapse of fusoid cells. (A) Adaxial (ad) and abaxial (ab) epidermis and vascular bundles (vb) differentiated (except the bulliform cells), and differentiation of ground meristem cells with fusoid cells readily distinguishable. (B) Several fusoid cells adjacent to the vascular bundles and inset showing a young fusoid cell with content. (C) Initial fusoid cell giving rise to a derivative fusoid cell (internal division within an initial fusoid cell). (D) Subsequent division from initial and derivative fusoid cells (internal divisions within an initial fusoid cell). (E) Vascular bundle between one (left) and three (right) fusoid cells, with nuclei visible in the fusoid cells. (F) Degradation of the middle lamella between fusoid and chlorenchyma cells. (G, H) Fusoid cell divisions accompanying vascular bundle maturation. Note the fully differentiated vascular tissue and enlarged bundle sheath cells in (G). (I) Degradation of the middle lamella between fusoid and chlorenchyma cells following bundle maturation. (J) Fusoid cell stretched and flattened along the lateral axis as a result of the degradation of the middle lamella and leaf blade enlargement. OBS: For additional information, see Supplementary Data S2. Scale bars (C, D, E, H) = 10 µm; (A) =15 µm; (I) = 30 µm; (F, G, J) = 40 µm; (B, inset) = 50 µm. Fig. 3. View largeDownload slide Cross-sections of leaf blades (TEM) at later stages of development with emphasis on the development of fusoid cells in Poaceae. (A–D) Parodiolyra micrantha (Bambusoideae); (E, F) Streptochaeta spicata (Anomochlooideae); (G) Pharus latifolius (Pharoideae). ad, adaxial epidermis; ab, abaxial epidermis; cc, chlorenchyma cells. White arrows show starch granules, red arrows show cell divisions and asterisks show the initial cavities formed from the degradation of the middle lamella. (A) Initial and the first derivative fusoid cells (internal division) showing nuclei and cytoplasmic content, large vacuoles and formation of a cell plate. (B) Fusoid cell showing an internal division, with initial formation of cell plate. (C) At least four derivative fusoid cells, with a large vacuole each, all within an initial fusoid cell; showing three nucleoli in the one visible nucleus. (D) Detail of a fusoid cell showing a cell plate, vacuoles and membranes from degraded organelles. (E, F) Fusoid cells with starch granules and initial degradation of the middle lamella. (G) Detail of starch granules in the fusoid cell. Scale bars: (G, H) = 1 µm; (B, I) = 3 µm; (D) = 3.5 µm; (C) = 4 µm; (A) = 5 µm. Fig. 3. View largeDownload slide Cross-sections of leaf blades (TEM) at later stages of development with emphasis on the development of fusoid cells in Poaceae. (A–D) Parodiolyra micrantha (Bambusoideae); (E, F) Streptochaeta spicata (Anomochlooideae); (G) Pharus latifolius (Pharoideae). ad, adaxial epidermis; ab, abaxial epidermis; cc, chlorenchyma cells. White arrows show starch granules, red arrows show cell divisions and asterisks show the initial cavities formed from the degradation of the middle lamella. (A) Initial and the first derivative fusoid cells (internal division) showing nuclei and cytoplasmic content, large vacuoles and formation of a cell plate. (B) Fusoid cell showing an internal division, with initial formation of cell plate. (C) At least four derivative fusoid cells, with a large vacuole each, all within an initial fusoid cell; showing three nucleoli in the one visible nucleus. (D) Detail of a fusoid cell showing a cell plate, vacuoles and membranes from degraded organelles. (E, F) Fusoid cells with starch granules and initial degradation of the middle lamella. (G) Detail of starch granules in the fusoid cell. Scale bars: (G, H) = 1 µm; (B, I) = 3 µm; (D) = 3.5 µm; (C) = 4 µm; (A) = 5 µm. Fig. 4. View largeDownload slide Mature leaf blades with emphasis on fusoid cells in Poaceae. LM (A–X) and CSEM (Y, Z). Cross-sections (A–J) show cavities (asterisks) resulting from the collapse of one to several fusoid cells (arrows). Longitudinal sections (K–R), paradermal sections (S–X) and cross-sections (Y–Z) show that each cavity is delimited by successive collapsed (rarely non-collapsed) fusoid cells arranged perpendicular to the veins along the lateral axis and along the proximo-distal axis of the entire lamina. (A, K, S) Streptochaeta spicata (Anomochlooideae). (B, L, M, N) Pharus latifolius (Pharoideae). (C, U) Guaduella oblonga (Puelioideae). (D) Luziola spruceana (Oryzoideae). (E, O) Oryza latifolia (Oryzoideae). (F) Parodiolyra micrantha (Bambusoideae). (G, Q, W) Aulonemia aristulata (Bambusoideae). (H, V) Otatea rzedowskiorum (Bambusoideae). (I) Glyceria striata (Pooideae). (J, R, X) Rugoloa pilosa (Panicoideae). (P) Raddia brasiliensis (Bambusoideae). (Y, Z) Guadua angustifolia (Bambusoideae). Scale bars: (Y, Z) = 6 µm; (W) = 12 µm; (P, R, X) = 25 µm; (M, N) = 30 µm; (K, L, O, Q, S, V) = 40 µm; (A–C, E–H) = 50 µm; (D) = 60 µm; (I, U) = 90 µm; (J) = 100 µm; (T) = 150 µm. Fig. 4. View largeDownload slide Mature leaf blades with emphasis on fusoid cells in Poaceae. LM (A–X) and CSEM (Y, Z). Cross-sections (A–J) show cavities (asterisks) resulting from the collapse of one to several fusoid cells (arrows). Longitudinal sections (K–R), paradermal sections (S–X) and cross-sections (Y–Z) show that each cavity is delimited by successive collapsed (rarely non-collapsed) fusoid cells arranged perpendicular to the veins along the lateral axis and along the proximo-distal axis of the entire lamina. (A, K, S) Streptochaeta spicata (Anomochlooideae). (B, L, M, N) Pharus latifolius (Pharoideae). (C, U) Guaduella oblonga (Puelioideae). (D) Luziola spruceana (Oryzoideae). (E, O) Oryza latifolia (Oryzoideae). (F) Parodiolyra micrantha (Bambusoideae). (G, Q, W) Aulonemia aristulata (Bambusoideae). (H, V) Otatea rzedowskiorum (Bambusoideae). (I) Glyceria striata (Pooideae). (J, R, X) Rugoloa pilosa (Panicoideae). (P) Raddia brasiliensis (Bambusoideae). (Y, Z) Guadua angustifolia (Bambusoideae). Scale bars: (Y, Z) = 6 µm; (W) = 12 µm; (P, R, X) = 25 µm; (M, N) = 30 µm; (K, L, O, Q, S, V) = 40 µm; (A–C, E–H) = 50 µm; (D) = 60 µm; (I, U) = 90 µm; (J) = 100 µm; (T) = 150 µm. Fig. 5. View largeDownload slide Cross-sections of Joinvillea ascendens (Joinvilleaceae) leaf blades (LM) showing the developmental stages of colourless cells (blue arrows). ab, abaxial; ad, adaxial; cc, chlorenchyma cells; vb, vascular bundle. Early stages of development (A–C) and maturity (D, E). (A, B, C) Subsequent developmental stages of colourless cells. (D) Major vascular bundle and chlorenchyma cells completely differentiated and colourless cells showing intact cell walls. (E) Colourless cells, always non-collapsed. OBS. The rosette-like inclusion observed in one of the colourless cells in (D) is a technical artefact caused by embedding and staining methods. Scale bars: (B–D) = 20 µm; (A) = 25 µm (A); (E) = 40 µm. Fig. 5. View largeDownload slide Cross-sections of Joinvillea ascendens (Joinvilleaceae) leaf blades (LM) showing the developmental stages of colourless cells (blue arrows). ab, abaxial; ad, adaxial; cc, chlorenchyma cells; vb, vascular bundle. Early stages of development (A–C) and maturity (D, E). (A, B, C) Subsequent developmental stages of colourless cells. (D) Major vascular bundle and chlorenchyma cells completely differentiated and colourless cells showing intact cell walls. (E) Colourless cells, always non-collapsed. OBS. The rosette-like inclusion observed in one of the colourless cells in (D) is a technical artefact caused by embedding and staining methods. Scale bars: (B–D) = 20 µm; (A) = 25 µm (A); (E) = 40 µm. Fig. 6. View largeDownload slide Cross-sections of leaf blades (LM) with no fusoid cells at early stages of development (A–D) and at maturity (E–I). (A–F) Setaria scabrifolia (Panicoideae). (G) Brachyelytrum erectum (Pooideae). (H) Centotheca lappacea (Panicoideae). (I) Flagellaria indica (Flagellariaceae). ad, adaxial epidermis; ab, abaxial epidermis; cc, chlorenchyma cells; vb, vascular bundles. Blue coloration highlights procambial strands (pr). (A) Early procambial strand of the midvein, protoderm (pt) and ground meristem (gm). (B) Midvein and initial development of minor procambial strands. (C) Subsequent development of procambial strands. (D) Major and minor procambial strands and initial differentiation of ground meristem cells into chlorenchyma cells. (E–I) Major and minor vascular bundles completely differentiated, as well as the chlorenchyma cells. OBS: For additional information, see supplementary note provided in the Supplementary Data S2. Scale bars: (A–D) = 13 µm; (F, H) = 40 µm; (E, I) = 50 µm; (G) = 100 µm. Fig. 6. View largeDownload slide Cross-sections of leaf blades (LM) with no fusoid cells at early stages of development (A–D) and at maturity (E–I). (A–F) Setaria scabrifolia (Panicoideae). (G) Brachyelytrum erectum (Pooideae). (H) Centotheca lappacea (Panicoideae). (I) Flagellaria indica (Flagellariaceae). ad, adaxial epidermis; ab, abaxial epidermis; cc, chlorenchyma cells; vb, vascular bundles. Blue coloration highlights procambial strands (pr). (A) Early procambial strand of the midvein, protoderm (pt) and ground meristem (gm). (B) Midvein and initial development of minor procambial strands. (C) Subsequent development of procambial strands. (D) Major and minor procambial strands and initial differentiation of ground meristem cells into chlorenchyma cells. (E–I) Major and minor vascular bundles completely differentiated, as well as the chlorenchyma cells. OBS: For additional information, see supplementary note provided in the Supplementary Data S2. Scale bars: (A–D) = 13 µm; (F, H) = 40 µm; (E, I) = 50 µm; (G) = 100 µm. Stage 1: Meristems and initial differentiation of cells into dermal and vascular tissues (LM) The initial stage of leaf-blade development is the same in all studied species and occurs from the base towards the apex (acropetally) and from the centre towards the margins (centrifugally) (e.g. Figs 1A–F and 6A–D). The differentiation of tissues may occur simultaneously, but often the protoderm (outermost meristematic layer) is the first meristem to become differentiated, followed by the procambium (innermost meristematic cells, usually with a large nucleus) and the ground meristem (all the remaining meristematic cells, between protoderm and procambium) (e.g. Figs 1A–F and 6A–D). Protodermal cells start anticlinal divisions to form a single stratum of epidermal cells (e.g. Fig. 1A, B). Simultaneously, procambial cells start anticlinal, periclinal and tangential divisions to give rise to vascular elements (e.g. Fig. 1A–C). Considering the centrifugal differentiation of tissues, the first major procambial strand will form the midvein (e.g. Fig. 1A), and then additional veins are formed by adjacent and also major procambial strands (e.g. Fig. 1B–F). Minor procambial strands are formed between pre-existing major ones as a result of leaf-blade enlargement (e.g. Fig. 1E). Mesophyll precursor cells from the ground meristem are mainly isodiametric and still undifferentiated at this point (e.g. Figs 1A–F and 6A, B), although some ground meristem cells are beginning their differentiation into mesophyll cells, in which cells below the epidermis and adjacent to vascular bundles start to divide mainly in the anticlinal and periclinal planes (e.g. Figs 1D, E and 6B). Some enlargement of the mesophyll cells destined to become fusoid cells may be observed at this stage (e.g. Fig. 1D–F). Stage 2: Ongoing differentiation of dermal, vascular tissues and ground meristem cells (LM) At this stage, the epidermis is completely differentiated (except the bulliform cells, which are large, thin-walled epidermal cells that occur in groups in the intercostal zone of the adaxial epidermis). The midvein and lateral vascular bundles are readily distinguishable, although the maturation of the conducting elements is still in progress (e.g. Fig. 2A–C). According to their position in the mesophyll, cells from the ground meristem will give rise to bundle sheath extensions. Ultimately comprising sclerenchymatous or parenchymatous cells, bundle sheath extensions are formed above and below at least the major vascular bundles, whereas isolated sclerenchymatous cells are mainly formed at the margins. Parenchyma cells that start to become heavily sclerified are observed adjacent to the bulliform cells and adjacent to the abaxial epidermis directly beneath the clusters of bulliform cells in A. aristulata (Poaceae, Bambusoideae) (Fig. 4G, mature leaf). Other mesophyll parenchymatous cells are formed primarily between vascular bundles and can be of six types according to the anatomical path of each species: (1) peg cells – thin-walled photosynthetic cells recognized by forming numerous symmetrical ‘arms’, irregularly spaced; (2) colourless cells – thin-walled cells adjacent to vascular bundles; (3) arm cells – thin-walled photosynthetic cells that exhibit asymmetrical cell wall invaginations (lobes) extending to different depths; (4) rosette cells – thin-walled photosynthetic cells that exhibit shallow cell-wall invaginations (lobes) around their periphery; (5) fusoid cells; and (6) irregular-shaped cells – thin-walled photosynthetic cells with no characteristic shape. Parenchymatous cells differentiate mainly into peg cells in F. indica (Flagellariaceae); irregular-shaped cells and colourless cells in J. ascendens (Joinvilleaceae); irregular-shaped or arm cells and fusoid cells in Streptochaeta spicata (Poaceae, Anomochlooideae) and Pharus latifolius (Poaceae, Pharoideae); irregular-shaped cells in Brachyelytrum erectum, Diarrhena obovata (Poaceae, Pooideae), Centotheca lappacea, Rugoloa pilosa and S. scabrifolia (Poaceae, Panicoideae), and irregular-shaped cells and fusoid cells in Glyceria striata (Poaceae, Pooideae); rosette cells in Ehrharta erecta (Poaceae, Oryzoideae); and arm cells or rosette cells and fusoid cells in A. aristulata, C. attenuata, Otatea rzedowskiorum and Raddia brasiliensis (Poaceae, Bambusoideae). These cell types exhibit contents visible at this stage and will form the chlorenchyma tissue (e.g. Fig. 2A–J). Irregular-shaped cells usually become arranged in a variable number of layers parallel to the epidermis, but in R. pilosa (Fig. 2A) and S. scabrifolia (Fig. 6D, E) they become radiate and surround the vascular bundles. In the members of Bambusoideae herein studied, arm cells usually become arranged into one or two layers of cells below the adaxial epidermis and rosette cells in just one layer above the abaxial epidermis (e.g. Fig. 2B), whereas in E. erecta (Oryzoideae) the number of layers of rosette cells is usually variable (not shown). During the differentiation of the chlorenchyma cells, the cells that occupy the central portion of the mesophyll, adjacent to each side of the vascular bundle, start to become distinguishable from the other parenchymatous cells and will form colourless cells in J. ascendens (Fig. 5A–C) and fusoid cells in the members of Poaceae that produce them (e.g. Figs 1E, F and 2A, B). This occurs in the same mesophyll region that differentiates the peg cells in F. indica (Fig. 6I, mature leaf) and irregular-shaped cells in Poaceae members with no development of fusoid cells (Fig. 6E–H, mature leaf). Stage 3: Final differentiation of tissues and the ongoing development of fusoid cells (LM and TEM) In J. ascendens, a single colourless cell (initial) divides to form three or four derivative cells (Fig. 5A–C); they then gradually increase in size along with the differentiation of the leaf blade and it is possible to observe the complete formation of their cell walls with LM (Fig. 5A–E). Fusoid cell differentiation in members of Poaceae herein studied occurs in much the same way; however, with LM we often observe the increase in size of the single fusoid cell, as in R. pilosa (Fig. 2A), A. aristulata (Fig. 2B inset), O. rzedowskiorum (Fig. 2E, H) and P. latifolius (Fig. 2G), with some content but no apparent cell divisions or, in the same sample in some taxa, cell divisions within the initial fusoid cell are clearly visible with LM [Fig. 2A (R. pilosa), Fig. 2C–E, H (O. rzedowskiorum) and Fig. 2F, G, I (P. latifolius)]. With TEM, we are able to easily observe division of the initial fusoid cell to form derivative cells, the vacuoles increasing in size with initial formation of cell plates, as in Parodiolyra micrantha (Fig. 3A–D), as well as in J. ascendens (not shown). A number of amyloplasts with starch grains in the developing fusoid cells are also often observed (e.g. Fig. 3E–G). At this point in time, peg cells, irregular-shaped cells and arm cells are almost all completely differentiated, and the rosette cells (abaxially positioned) in members of Bambusoideae (Poaceae) studied here become topographically distinct from the arm cells (e.g. Fig. 2B). Except for the fusoid cells, mesophyll differentiation often occurs simultaneously, but there are temporal differences in reaching complete maturity depending on the developmental characteristics of each species (e.g. earlier or later differentiation of mesophyll cells in relation to fusoid cell enlargement). Fusoid cells of adjacent bundles are virtually always separated by one or maybe two rosette cells in Bambusoideae members. Along with leaf enlargement, there is degradation of the middle lamella between adjacent fusoid cells within the same proximo-distal row and some chlorenchyma cells, and the fusoid cells are stretched and flattened along their long axis (along the lateral axis towards both margins of the leaf blade), as in P. latifolius (Fig. 2F, I, J). Stage 4: Final development of fusoid cells and mature leaves (LM and CSEM) Figure 4A–X shows many LM images of mature leaf blades of Poaceae members with fusoid cells, organized according to the most recent phylogenetic classification by Soreng et al. (2017), and two CSEM images of the mature leaf blade of G. angustifolia (Bambusoideae, Poaceae) mature leaf blade (Fig. 4Y, Z). Peg cells in F. indica (Fig. 6I), irregular-shaped cells in S. spicata (Fig. 4A), P. latifolius (Fig. 4B), Pooideae members (e.g. Fig. 4I, G. striata) and Panicoideae members (e.g. Fig. 4J, R. pilosa; Fig. 6E, S. scabrifolia), rosette cells in Oryzoideae members (e.g. Figs. 4D, Luziola spruceana; 4E, Oryza latifolia) and Bambusoideae members (e.g. Fig. 4F, P. micrantha; Fig. 4G, A. aristulata; Fig. 4H, O. rzedowskiorum), arm cells in Bambusoideae members (e.g. Fig. 4F, P. micrantha; 4G, A. aristulata; 4H, O. rzedowskiorum) and the colourless cells between vascular bundles (usually three or four) in J. ascendens (Fig. 5D) are completely differentiated (note that to better illustrate mature mesophyll cells, we included complementary images from species for which a developmental study was not herein performed). Yet, along with the breakdown of the middle lamella during leaf enlargement, fusoid cells (including any derivatives that may be present) gradually collapse along their long axis, leaving a cavity between adjacent successive fusoid cells (e.g. Fig. 4K–X, asterisks). In mature leaf blades, the fusoid cell in cross-section is a usually a cigar-shaped cavity (e.g. Fig. 4A–J, asterisks), and, due to the collapse, forms an I-shaped cell in longitudinal and paradermal views (e.g. Fig. 4K–Z, arrows). However, longitudinal and paradermal sections reveal that not all fusoid cells collapse (e.g. Fig. 4M–O, Q, R, T, W), since their cell walls still have a contact zone with the bundle sheath and other parenchymatous mesophyll cells (Fig. 4K–X, Supplementary Data Video). Also, as seen in paradermal view, fusoid cells comprising one longitudinal row of cells do not touch fusoid cells from any other row; however, they touch chlorenchyma cells and vascular sheath cells, and may touch adjacent fusoid cells within the same row if not collapsed (e.g. Fig. 4T–W, Supplementary Data Video) (note that ‘row’ refers to a set of many fusoid cells adjacent to each other along the entire lamina). DISCUSSION Fusoid cell origin and development Mesophyll with large colourless cells has been reported in Joinvilleaceae (Bayer and Appel, 1998), a family that currently comprises only two known species (J. ascendens and Joinvillea elegans). In this family, it is curious that colourless cells occur in the same mesophyll region as fusoid cells in the Poaceae, adjacent to and between vascular bundles. Also, we herein observed that the origin of a fusoid cell is from the ground meristem and thus is the same as a colourless cell in J. ascendens, even though there is developmental variation worth taking into account. In J. ascendens, a single colourless cell (initial) divides to form its derivatives and, as leaf maturation proceeds, initial and derivative cells do not collapse, remaining intact until their complete development. In the Poaceae species herein studied, however, the development of a fusoid cell occurs mostly in the same way as a colourless cell, but the initial and its derivative cells (internal divisions from a single fusoid cell with cell plates only) often collapse perpendicularly to the proximo-distal axis of the leaf blade later in their development, leaving cigar-shaped cavities in the mesophyll as seen in cross-section. Hence, the development of colourless and fusoid cells is quite similar and, considering their same topography and ground meristem origin, they are clearly homologous, as well as being homologous to the mesophyll cells adjacent to the vascular bundles in those species with no colourless or fusoid cells. Also, of note, colourless cells are more identifiable only when the vascular bundles are well differentiated. It is noteworthy that our results show that the origin of a fusoid cell is always from the ground meristem and that fusoid cells occur in seven subfamilies of the Poaceae, ubiquitously in the early-diverging lineages (Anomochlooideae, Pharoideae and Puelioideae), within the BOP clade (Oryzoideae, virtually all Bambusoideae, and Pooideae) and sporadically in the Panicoideae (PACMAD clade). Within the latter subfamily, the meristematic origin of the fusoid cells in Rugoloa (previously placed in Panicum) was thought to be distinct from the origin of fusoid cells in the early-diverging lineages and the BOP clade (Grass Phylogeny Working Group, 2001—the BOP clade referred to as the BEP clade in this work). In that work, these cells were assumed to be derived from laterally extended bundle sheaths and thus were referred to as fusoid-like cells. However, the homologies herein observed demonstrate that there is no reason to apply distinctive terminology for these cells in the Panicoideae. According to Sugimoto et al. (2011), transdifferentiation encompasses the process by which cells transform into another cell type outside of their already established differentiation paths. In contrast, a putative fusoid cell is clearly distinguishable from any other mesophyll cell even at the earliest stages and throughout development until its complete differentiation. The most recent work on the development of fusoid cells (in two species of Guadua) reported evidence of transdifferentiation of chlorenchyma cells into fusoid cells (Vega et al., 2016); however, the developmental work herein performed shows no evidence of transdifferentiation for the 13 species we sampled across the grass family. Vega et al. (2016) also described a conspicuous cell wall invagination during the differentiation of chlorenchyma cells into fusoid cells, which likewise was not herein observed. Since the cell wall invagination reported in that work is also evidenced in bundle sheath cells (see Fig. 1B, C in Vega et al., 2016), which do not have any lobes at maturity, it is very likely that the observed invaginations are a technical artefact caused by fixative methods. Of note, we had similar results in materials pre-fixed for <48 h. What appears to be a mature fusoid cell in leaf-blade cross-sections is herein interpreted as a cavity delimited by successive collapsed fusoid cells arranged perpendicularly to the veins (i.e. perpendicular to the proximo-distal axis in the lateral plane), with the caveat that sometimes fusoid cells do not collapse. A similar interpretation was given by Vega et al. (2016), referring to these apparent ‘cells’ as intercellular gas spaces delimited by successive collapsed fusoid cells. We consider ‘cavity’ to be the appropriate term because many Poaceae species have intercellular gas spaces in the mesophyll (not necessarily only as seen in cross-section and not in this configuration; e.g. Dengler et al., 1994; Gielwanowska et al., 2005), and thus it distinguishes more clearly between the types of intercellular spaces in order to avoid possible misinterpretation. Since fusoid cells collapse perpendicularly to their long axis but keep themselves partially connected to mesophyll cells adjacent to the epidermis and vascular sheath cells, they typically form cavities in the mesophyll rather than a complex network of small intercellular gas spaces. We propose that a single cavity as seen in a cross-section of a mature leaf blade is typically the result of the development and collapse of several fusoid cells (the initial and its internal derivative cells). During leaf development, we are not often able to perceive fusoid cell division with LM, and thus each of the successive I-shaped collapsed fusoid cells delimiting the cavities has always been interpreted as resulting from the collapse of a single fusoid cell. This is because the cell wall does not reach its full formation between derivative fusoid cells, often forming only cell plates (early stage), which is really difficult to observe with LM. Light microscopy techniques were able to clearly show internal fusoid cell divisions just in S. spicata (Anomochlooideae) and P. latifolius (Pharoideae); however, the division of a fusoid cell initial and its derivatives was observed with TEM techniques in all species herein studied that exhibit fusoid cells. Nonetheless, we cannot exclude the possibility that some initial fusoid cells simply enlarge and never divide internally before they collapse. In contrast, adjacent to the midrib, full formation of the cell walls of derivative cells does occur, albeit rarely, and thus several fusoid cells may be observed in this position in cross-section, as reported in mature leaves of Fargesia yunnanensis and Dendrocalamus giganteus (Wang et al., 2016). As noted by Ellis (1976, p. 105), due to the collapse of fusoid cells, longitudinal and paradermal sections of the leaf blade show these cells to have very narrow lumina in cross-section, having themselves collapsed in such a way that the tissue resembles a row of many ‘I’s’, which gives a lacunar aspect to the mesophyll (Jacques-Félix, 1962). We follow Ellis (1976) in describing these collapsed cells as I-shaped, even though they exhibit variation and can even appear dumbbell-shaped in some cases (e.g. Fig. 4W). As a final remark on development, the gradual collapse of fusoid cells observed in all studied species strongly suggests that they exhibit programmed cell death (PCD), as previously suggested for some Bambusoideae species (Vega et al., 2016; Wang et al., 2016). Quite often there is no evidence of a nucleus even at earlier stages of development as seen with LM, which implies an earlier PCD process, as previously suggested for F. yunnanensis (Wang et al., 2016). We will discuss PCD in more detail in a subsequent section. Phylogenetic implications of fusoid cells for the graminid clade The homology herein observed between the colourless cells in Joinvilleaceae and the fusoid cells in Poaceae is consistent with the relationships within the graminid clade as derived from molecular data (Givnish et al., 2010; Bouchenak-Khelladi et al., 2014). Within Flagellariaceae, which is sister to the rest of the graminid clade, there is no evidence of differentiated colourless or fusoid cells in the mesophyll. The Ecdeiocoleaceae do not produce developed leaves (Linder et al., 1998; Briggs, 2011), but regardless of whether it is sister to Joinvilleaceae or to Poaceae, the most parsimonious explanation is that the cell type ancestral to both colourless and fusoid cells evolved along the stem of the [(Joinvilleaceae + Ecdeiocoleaceae) + Poaceae] or [Joinvilleaceae + (Ecdeiocoleaceae + Poaceae)] clade. Further anatomical, developmental and molecular studies are needed to better document the origin of the fusoid cells in the graminid clade. The first attempt to produce a phylogenetic hypothesis of relationships within Poaceae was based exclusively on morphological data, in which Bambusoideae (including herbaceous tribes such as Anomochloeae, Phareae and Streptochaeteae) was interpreted as monophyletic based on the presence of arm cells and fusoid cells (Kellogg and Campbell, 1987). Clark et al. (1995), based on a family-wide analysis of plastid ndhF sequence data, resolved the three tribes mentioned above as forming two of the three early-diverging lineages within the family, with a more narrowly circumscribed Bambusoideae placed in the BOP clade. They therefore concluded that the presence of fusoid cells, a character previously useful for delimiting Bambusoideae, was probably a synapomorphy for Poaceae. A broader phylogenetic analysis of the Poaceae, incorporating both molecular sequence and morphological data, was elaborated by the Grass Phylogeny Working Group (2001) and clearly showed the fusoid cells as plesiomorphic within the family, but also typically found in Bambusoideae species and Streptogyna, then of uncertain placement in the BOP clade. Our results confirm the plesiomorphic presence of fusoid cells within Poaceae. As previously mentioned, fusoid cells occur in all three early-diverging lineages (Anomochlooideae, Pharoideae and Puelioideae), unevenly within the BOP clade (almost all Bambusoideae, but only in some Oryzoideae, and Pooideae), and in a few Panicoideae species. In the BOP clade, Oryzoideae is sister to Bambusoideae + Pooideae, and within Oryzoideae the tribe Streptogyneae is sister to the remainder of the subfamily: Ehrharteae, Oryzeae (including the subtribes Oryzinae and Zizaniinae) and Phyllorachideae (Soreng et al., 2015, 2017). Within Oryzoideae, fusoid cells are known only in the early-diverging Streptogyna (Streptogyneae) (Soderstrom et al., 1987) and in some species within the tribe Oryzeae, subtribe Zizaniinae (Tateoka, 1963; Kellogg, 2015; Leandro et al., 2016a) and subtribe Oryzinae (Oryza, this study). Within Pooideae, fusoid cells are reported only in Brachyelytrum (Barkworth et al., 2007; Stephenson and Saarela, 2007), both works, however, only mentioning (not accompanied by anatomical images) the presence of fusoid cells in the monotypic Brachyelytreae (herein not observed in B. erectum but documented in G. striata). Since the lack of fusoid cells in Brachyelytrum has also been reported in the literature (e.g. Tateoka 1957 – accompanied by a drawing of the leaf blade in cross-section; Campbell et al., 1986; Watson et al., 1992 onwards; Kellogg, 2015 – these latter three works also only mentioning the lack), the occurrence of fusoid cells in this genus is surely controversial and needs further investigation. In addition, for Glyceria, the occurrence of fusoid cells could be supported by the record of the sporadic presence of large cavities in the mesophyll of some species (Watson et al., 1992 onwards), which probably was interpreted in the same way as the mesophyll cavities observed, for instance, in the former Panicum sect. Laxa (Killeen and Clark, 1986), herein confirmed as being fusoid cells in R. pilosa (formerly Panicum pilosum). Hence, the presence of the fusoid cells in the early-diverging lineages, Streptogyneae, Zizaniinae, Oryziinae, a few Pooideae and generally in the Bambusoideae, and their lack in Ehrharteae, Phyllorachideae and most Pooideae clearly suggest multiple losses within the BOP clade (Kellogg, 2015). The same developmental origin of the fusoid cells herein confirmed between Panicoideae species and other Poaceae members is also relevant to phylogenetic inferences within the family. Fusoid cells have been reported in a few species of Rugoloa, Dallwatsonia (Killeen and Clark, 1986; Zuloaga et al., 1992 – both genera referred to as Panicum in this work), Homolepis (Watson et al., 1992 onwards) and Canastra (Morrone et al., 2001). These genera are all classified within the tribe Paspaleae, which, along with Paniceae and several other tribes with no fusoid cells reported, comprise the subfamily Panicoideae (Soreng et al., 2015, 2017). Thus, fusoid cells were probably lost early in the evolution of the PACMAD clade but regained in the Paspaleae. A broader sampling within Panicoideae as well as a formal character optimization of the presence of fusoid cells using the most recent molecular phylogeny of the family are needed to verify the evolution of this character within Poaceae. Some evolutionary and functional insights on fusoid cells based on TEM data Several possible functional roles for fusoid cells in mature leaves have been proposed, and can be summarized as structural (Vega et al., 2016), light scattering (March and Clark, 2011), photorespiration-related (Clark, 1991; Wang et al., 2016) and water dynamics-related (Vieira et al., 2002; Wang et al., 2016). These functions may not necessarily be mutually exclusive and thus may occur simultaneously in mature leaves. Hence, in this section we informally correlate these findings with environmental characteristics and our developmental results from young leaf blades. Estimates of divergence times for lineages within Poales vary, but there is agreement that Poales initially evolved mainly in open habitats and that the earliest transitions to shade habitats occurred in the graminid clade (Givnish et al., 2010; Bouchenak-Khelladi et al., 2014). The early-diverging lineages of Poaceae comprise plants growing in shaded forests or along forest margins, and more recent phylogenetic analyses based on plastomes suggest that the origin and early diversification of the BOP clade were associated with forested habitats as well (Grass Phylogeny Working Group II, 2012; Burke et al., 2016a, b). The presence of fusoid cells in the early-diverging grass lineages plus Streptogyneae and Bambusoideae suggests the correlation of these cells with shaded environments, although some forest grass lineages lack fusoid cells (e.g. Phyllorachideae of the Oryzoideae, and Diarrhena and apparently Brachyelytrum of the Pooideae in the BOP clade). In contrast, the presence of fusoid cells in some other Oryzoideae, comprising plants growing in open and often wet environments, suggests the influence of other environmental factors. The pattern with regard to habitat and the origin and diversification of the PACMAD clade is more ambiguous, even if the Panicoideae are inferred as the sister to the remainder of the clade (Burke et al., 2016b; Teisher et al., 2017). However, many early-diverging Panicoideae are associated with forest habitats but do not have fusoid cells (e.g. Centotheca, this study), although the facultative occurrence of fusoid cells has been reported in this genus (Watson et al., 1992 onwards, not accompanied by anatomical images), as well as lateral bundle sheath extensions in some centothecoid taxa (e.g. Soderstrom and Decker, 1973, also without any anatomical images), and at least some of the Paspaleae reported to have fusoid cells are found in open habitats (e.g. Canastra; Morrone et al., 2001). Since the facultative occurrence of fusoid cells has been reported in other grass species (e.g. Metcalfe, 1956; Wu, 1962; Pearson et al., 1994) and there is evidence of fusoid cells occurring in species associated with shaded and sunny habitats (e.g. March and Clark, 2011; Leandro et al., 2016a; Morrone et al., 2001), the occasional retention of fusoid cells in, for instance, Centotheca and Brachyelytrum would not be surprising but remains to be investigated. This background is important in understanding the potential roles played by the fusoid cells. Our developmental results show fusoid cells, which begin to enlarge and differentiate before the chorenchyma cells, with no chloroplasts but with several amyloplasts per fusoid cell, while the fusoid cells are still intact. Plants living in shaded environments often have wide leaves (Esau, 1977; Craine, 2009), presumably to maximize light absorption, which optimizes their survival under low levels of light. In this kind of environment, in general, there is suppression of photorespiration, with stomatal closure and consequent increase in plant growth (Walters and Reich, 1999; Craine, 2009). Thus, considering the low rates of photorespiration in shaded environments, along with our results showing no evidence of starch storage in the differentiating chlorenchyma cells, we argue that, at early stages of development, fusoid cells could act in the synthesis and storage of starch granules. In addition, the presence of plasmodesmata between chlorenchyma cells and fusoid cells, although few in number, supports the possibility of glucose transport from photosynthetic cells to fusoid cells, even though, according to Wang et al. (2016), no plasmodesmata or starch granules were associated with fusoid cells in F. yunnanensis. Of note, in this study, plasmodesmata in fusoid cells are observed only at earliest stages of mesophyll development. Further ultrastructural and functional studies should assist in the interpretation of the relationship of CO2 supply to the potential function of fusoid cells early in development. As previously mentioned, fusoid cell collapse (and apparent death) is often observed in mature leaves. At early stages of development, our TEM analysis reveals vacuoles increasing in size, internal division with initial formation of cell plates, and remains of membranes resulting from the lysis of organelles. Since most of these characteristics are typically described in plant cells undergoing PCD (Zhou et al., 2009), it is conceivable that the ultimate fate of these cells is determined by a PCD process, as previously suggested (Vega et al., 2016; Wang et al., 2016). Although our results support the presence of a PCD process during the development of fusoid cells, further investigation is needed to verify the occurrence of such a process. Conclusions During leaf differentiation, a single fusoid cell (initial) originating from the ground meristem may divide internally to form several fusoid cells (derivatives), although with formation of cell plates only. Our evidence suggests that this internal division occurs in many but not necessarily in all fusoid cells of a given leaf. In mature leaf blades as seen in cross-section, a fusoid cell is actually usually a cavity resulting from the collapse of the larger initial fusoid cell and its derivatives, most likely through a PCD process. Each cavity is delimited by successive collapsed fusoid cells arranged perpendicularly to the veins (i.e. perpendicular to the proximo-distal axis in the lateral plane) and along the entire lamina. Considering that they share the same topography and ground meristem origin, fusoid cells in Poaceae and colourless cells in Joinvilleaceae are homologous. Also, the meristematic origin of fusoid cells in Panicoideae is the same as in the early-diverging lineages and BOP clade (Poaceae), and thus they are homologous within the family. In addition, the presence of fusoid cells in the early-diverging lineages, Streptogyneae, Zizaniinae, a few Pooideae and Paspaleae and generally in the Bambusoideae, and their lack in the Ehrharteae, Phyllorachideae, Oryzinae and most Pooideae and Panicoideae, clearly suggest multiple losses within the BOP clade and an ancestral loss in the PACMAD clade with at least one regain in the Paspaleae (Panicoideae). We hypothesize that one role played by the fusoid cells is related to synthesis and storage of starch granules at early stages of leaf development. Further study is needed to verify functional analogies since this foundation is necessary to understand the evolution of mesophyll cells within the graminid clade. SUPPLEMENTARY DATA Supplementary data are available online at https://academic.oup.com/aob and consist of the following. S1: phylogenetic classification of Poaceae redrawn based on Soreng et al. (2015, 2017). S2: developmental stages of Aulonemia aristulata (Döll) McClure. Video: showing a tridimensional reconstruction of mature fusoid cells. ACKNOWLEDGEMENTS This research was completed as a partial fulfilment for the first author’s scholarship funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES (PDSE grant number 99999.003340/2015-05) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq (GD grant number 163550/2012-3; PDJ grant number 150797/2017-6 ). V.L.S. was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq (grant number 301692/2010-6). Field work, TEM and CSEM were supported by National Science Foundation grants DEB-1120750 and DEB-1342787 to L.G.C. The authors are grateful to Dr Harry T. 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