Introduction

The subfamily Oryzoideae (syn. Ehrhartoideae) is monophyletic and a member of the BOP clade [Bambusoideae + Oryzoideae + Pooideae] of grasses (Grass Phylogeny Working Group (GPWG) II 2012; Soreng et al. 2015). Oryzoideae (excluding Streptogyneae) share a combination of characters including spikelets with two glumes (developed to reduced), one or two sterile lemmas, and a single fertile floret (whether bisexual or unisexual) (GPWG 2001; Terrell et al. 2001).

Oryzeae is one of four tribes classified within Oryzoideae and comprises 13 genera (Soreng et al. 2015) with about 70 species that occur mostly in aquatic environments. Currently, Oryzeae is subdivided into two subtribes (Oryzinae and Zizaniinae) based on morphological, anatomical, embryological and molecular analyses (Duvall et al. 1993; Soreng et al. 2015). Zizaniinae (syn. Luziolinae—Terrell and Robinson 1974) comprises nine genera, including Luziola Juss.

Luziola consists of nine aquatic, monoecious, perennial species, distributed from North America to Argentina, with its center of diversity in South America (Swallen 1965; Terrell and Duvall 2000), where all nine species occur in Brazil (Filgueiras et al. 2015).

The Pantanal is characterized by a heterogeneous vegetation community, which is shaped by periodic flooding (Adámoli and Pott 1996), where five Luziola species exhibit important ecological adaptations to the periodic flooding typical of this tropical wetland. Luziola bahiensis (Steud.) Hitchc. is considered an occasionally aquatic species (Albuquerque 1981) which is shade tolerant and highly sensitive to drought and fire (Pott and Pott 2000). Also, Luziola bahiensis as well as L. peruviana Juss. ex J.F.Gmel. are invasive to native populations of rice (Oryza glumaepatula Steud. and O. latifolia Desv.) (Kissmann 1997). Luziola fragilis Swallen and L. peruviana are generally found in standing water, mainly in the ponds (“baías”), temporary ponds, and floodable grasslands (Pott and Pott 2000). Furthermore, Luziola peruviana may establish as emergent, floating or submerged, while L. fragilis usually colonizes areas impacted by anthropogenic activities, such as borrow pits—common in the Pantanal for water storage during the dry seasonal (Pott and Pott 2000). Luziola spruceana Benth. ex Doell and L. subintegra Swallen resemble native rice when in the vegetative stage, and are usually dispersed by floating mats (“baceiros”) when seasonal streams are present (Pott and Pott 2000).

The strongly similar morphological features of Luziola species in the vegetative stage raised the need for systematic and phylogenetic studies to the genus (Martinez-y-Perez et al. 2006a, b, 2008). Although surveys of anatomy of the leaf blade have revealed the utility of features in the delimitation of Poaceae (e.g., Metcalfe 1960; Tateoka 1963; Vieira et al. 2002; Guglieri et al. 2008; Pelegrin et al. 2009; Jesus-Junior et al. 2012), there is a dearth of information particularly among morphologically similar grasses in the vegetative stage.

Due to the morphological affinity and importance of correct delimitation of Luziola species in the vegetative stage, the aim of this work was to study leaf blade micromorphology and anatomy of Luziola bahiensis, L. fragilis, L. peruviana, L. spruceana and L. subintegra, effectively mapping useful features for the identification of taxa and to contribute towards future anatomical characterization of the genus.

Materials and methods

Sampling

The following five species of Luziola from the Pantanal, Brazil were studied: L. bahiensis, L. fragilis, L. peruviana, L. spruceana and L. subintegra. Due to the difficulty in finding reproductive stages in the field, for this work we studied only dry material from the CGMS herbarium collection to ensure the species identification (Table 1). Also, since Luziola species occur in environments with seasonal variation in water availability, this study was carried out with at least three specimens for each species (when possible) collected during different growing seasons (dry, moist or flood conditions). Our sampling was limited to the five species in the State of Mato Grosso do Sul because a parallel study was being carried out for the other four species of Luziola in the State of Bahia (K. R. B. Leite personal comm.).

Table 1 Species and vouchers for the micromorphological and anatomical studies of Luziola
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Leaf blade analysis

For SEM description of the silicified structures (trichomes and silica bodies), one fragment of approximately 0.5 cm2 of the middle portion of the leaf blade from the third stem node was removed from dry material. The fragment was carbonized at 200 °C, washed with sulphuric acid, later rinsed with distilled water to eliminate formed chloride and then, left to dry at room temperature (Figueiredo and Handro 1971). This dried material was also carbonized (at 800 °C). The resulting powder from the incineration was fixed in a cylindrical sample holder (stub) and coated with a thin sample of gold (Denton vacuum Desk III). For SEM description of the leaf blade surface, two fragments of approximately 0.5 cm2 of the same portion of the leaf blade representing adaxial and abaxial surfaces were removed from dry material, fixed on stubs, and also coated with a thin sample of gold. Observation and images were obtained with the aid of a scanning electron microscope (JEOL JSM-6380LV).

For anatomical study, fully expanded leaf blades were taken from the third stem node and sectioned at the middle portion. Each sample was immersed in a rewetting solution containing distilled water, ethanol, glycerin, and neutral detergent (Guglieri et al. 2008). The mixture was placed in an incubator at 60 °C for 3 days, after which samples were removed and stored in 70 % alcohol. The sample pieces were placed in polyethylene glycol 1.500 solution (adapted from Richter 1985) and cross and longitudinal sections were made using a Leica RM 2145 rotary microtome. The sections were diaphonized in 50 % sodium hypochlorite, rinsed in distilled water, and stained in Astra Blue and Safranin (Roeser 1972). Semi-permanent slides were mounted in 50 % glycerin, and analyzed under a light microscope (Olympus CX-41F).

Photomicrographs were obtained with the aid of the microscope (Leica DM 5500B) through the Leica Suite Application 3.8.0 software. The terminology for the leaf blade in cross section and in surface view primarily followed Ellis (1976, 1979), with subsequent updates to mesophyll terminology in Poaceae (Sanchez-Ken et al. 2001).

Results

Intraspecific anatomical variation, such as the area of the air gaps, tissue lignification, and trichome density were found for all species studied. Despite these variations, there are no intraspecific interference effects to identify these species, since all exhibit similar intraspecific micromorphological and anatomical characteristics. The main features of the micromorphology and anatomy of the leaf blade, useful to identification and delimitation of studied Luziola species, are summarized in Table 2.

Table 2 Main micromorphological and anatomical features of the leaf blade distinguishable among the studied Luziola species
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Micromorphology (SEM)

In surface view, the epidermis of all species has alternating long and short cells. The long cells are tabular shaped in the costal zone and mostly hexagonal in the intercostal zone (Figs. 1, 2). These cells present silicified outer walls with conical papillae densely covered by epicuticular rod-shaped wax, as in Luziola bahiensis (Figs. 1a, b, 2a). Short cells that develop as silica cells have dumbbell (Figs. 1b, 2c) or cross-shaped silica bodies (Fig. 1c, f–i, 2g–h, k–l). Dumbbell silica bodies are inserted parallel to the long axis of the leaf as seen in Luziola bahiensis (Fig. 1b). On the other hand, Luziola fragilis (Figs. 1d, 2g), L. peruviana (Figs. 1f, 2h), L. spruceana (Figs. 1g–h, 2k), and L. subintegra (Figs. 1i, 2l) have cross-shaped silica bodies that are inserted perpendicular to the long axis of the leaf blade (Table 2). Luziola fragilis (Fig. 1c) and L. peruviana (Fig. 1e) have epidermal furrows in the adaxial surface (Table 2).

Fig. 1
figure 1

Micromorphology of the leaf blade of Luziola species in surface view. Adaxial surface (a, c, e, g, i), abaxial surface (b, d, f, h). a, b L. bahiensis; c, d L. fragilis; e, f L. peruviana; g, h L. spruceana; i L. subintegra. CZ costal zone, IZ intercostal zone, Pr prickle hair, SB silica bodies, UT unicellular trichome. Arrows the stomata. Scale bars 100 μm

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Fig. 2
figure 2

Micromorphology of the leaf blade of Luziola species. Adaxial surface (a, f), abaxial surface (b) and silica bodies (c, g, h, k, l). a L. bahiensis; b L. fragilis; c L. bahiensis; d L. peruviana; e L. subintegra; f, g L. fragilis; h L. peruviana; i and j L. fragilis showing the comparison of the leaf blade margins; k L. spruceana; l L. subintegra. Pr prickle, SB silica bodies, UT unicellular trichome. Arrow stomata and asterisk conical papillae with epicuticular rod-shaped wax. Scale bars 2 μm (a, c, g, h, k, l); 10 μm (b); 50 μm (f, i, j)

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Stomata are paracytic with subsidiary cells which are low dome-shaped forming a narrow stomatal complex (e.g., Fig. 2b, d). Trichomes can be of three types: unicellular silicified with a (1) pointed or (2) rounded apex, and (3) prickles. The pointed trichomes occur on the adaxial surface in L. bahiensis (Fig. 1a), adaxial and abaxial surfaces in L. fragilis (Figs. 1c, d, 2b, f, i) and the adaxial surface in L. subintegra (Fig. 1i), while the rounded-apex trichomes occur on the adaxial and abaxial surfaces of L. fragilis (Fig. 1d) and on the abaxial surface of L. spruceana (Fig. 1h). Prickle hairs, short and silicified unicellular trichomes (Fig. 2e), are observed in the costal zone of both surfaces of the leaf blade in most species (e.g., Fig. 1f–g), except in L. fragilis (Fig. 1d) and L. spruceana (Fig. 1h), where they occur only on the abaxial surface. Epidermal cells form vertically elongated outgrowths which partially cover the pointed trichomes in L. fragilis (Figs. 1c, d, 2f, i; Table 2) and the rounded trichomes in L. spruceana (Fig. 1h).

With regard to the leaf blade margins, only Luziola fragilis has micromorphological dimorphism within the same sample, where pointed trichomes may be present (Fig. 2i) or absent (Fig. 2j), as well as vertical outgrowths (Fig. 2i).

Cross section (light microscopy)

The epidermis in cross section of all species consists of a single stratum of cells, with visible thickening of the external periclinal wall (Fig. 3). Along both surfaces conical papillae are noticeable (Figs. 3f–h, k–n, 4). All species possess amphistomatic leaf blades with associated substomatal chambers (Fig. 3k–n, asterisks). In both L. fragilis (Fig. 3b) and L. peruviana (Fig. 3d) the adaxial stomata occur in epidermal furrows. Stomata are mainly situated slightly below the epidermal surface (Fig. 3k–n) in all species. Fan-shaped arrays of bulliform cells are situated only in the adaxial surface above the level of other epidermal cells in all species (Fig. 3k–n).

Fig. 3
figure 3

Leaf blade anatomy of Luziola species. Cross section (a–h, k–n) and longitudinal section (i, j). a L. bahiensis; b L. fragilis; c L. spruceana; d L. peruviana; e L. subintegra; f L. bahiensis; g L. spruceana and h L. subintegra both showing superposed vascular bundles in the mesophyll and detail of the vascular bundle adjacent to fusoid cells; i L. spruceana; j L. subintegra; k L. bahiensis; l L. fragilis; m L. peruviana; n L. spruceana. AG air gap, FC fusoid cells, UT unicellular trichome. Arrowheads in (f, g, h) indicate silica bodies. Scale bars 10 μm (k, l, m, n); 25 μm (g detail, h detail); 50 μm (a, b, d, f–j); 100 μm (c, e)

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Fig. 4
figure 4

Anatomy of the leaf blade margin of Luziola species in cross section. Acute margins (a–c), obtuse margins (d). a, b L. fragilis; c L. spruceana; d L. subintegra. Scale bars 25 μm

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The mesophyll is formed mainly by rosette cells containing chloroplasts and vascular bundles (Fig. 3a–n). The rosette cells are lobed mostly at the same depth all the way around, mostly oriented horizontally to the epidermis (Fig. 3a–h) and radially around the vascular bundles, more visibly so in L. spruceana (Fig. 3g) and L. subintegra (Fig. 3h). Fusoid cells occur in L. spruceana (Fig. 3g, g detail, i) and L. subintegra (Fig. 3h, h detail and j), between vascular bundles and adjacent to the chlorenchyma cells (Table 2).

Vascular bundles are collateral, with conducting elements surrounded by a double sheath, the inner mestome sheath (pericyclic) and the outer parenchymatous sheath (endodermis) (e.g., Fig. 3a). All species have both first- and third-order vascular bundles in the mesophyll, as in L. bahiensis (Fig. 3f). Luziola spruceana (Fig. 3g) and L. subintegra (Fig. 3h) possess, in the midrib and throughout the leaf blade, sets of a second-order vascular bundle opposed to a first-order one (Table 2) both sharing the same parenchymatous sheath. All the sets of first- and third-order vascular bundles have adaxial and abaxial girders, usually abaxially interrupting the parenchymatous sheath, as seen in Luziola bahiensis (Fig. 3f).

The midrib in most species is distinguished by the presence of an abaxial keel (Fig. 3a, c–e), whereas a flat midrib is seen only in L. fragilis (Fig. 3b; Table 2). The number of bundles comprising the midrib varies, with two in L. bahiensis (Fig. 3a), L. fragilis (Fig. 3b) and L. peruviana (Fig. 3d) and several vascular bundles of different sizes in L. spruceana (Fig. 3c) and L. subintegra (Fig. 3e; Table 2). In this region, three and five vascular bundles are superposed in a column in L. spruceana (Fig. 3c) and L. subintegra (Fig. 3e), respectively. The other three species have two superposed vascular bundles in the midrib, as in L. fragilis (Fig. 3b). Air gaps occur in the midrib of most species, except L. fragilis (Fig. 3b) and L. peruviana (Fig. 3d).

The leaf margins are acute for most of the species (e.g., Fig. 4a–c), except in L. subintegra which possesses obtuse margins (Fig. 4d; Table 2). All species exhibit thickened epidermal cells in this region and sclerenchyma fibers below the epidermis (Fig. 4). Dimorphism of the leaf blade margin within the same sample was also observed in the cross sections of L. fragilis, mainly concerning the thickness and number of the sclerenchyma cells (Fig. 4b, c).

Discussion

Some observed features in Luziola species are common for Oryzoideae, such as the epidermis with silicified outer walls and wax impregnation, fan-shaped arrays of bulliform cells on the adaxial surface, collateral vascular bundles surrounded by a double sheath, and long cells with multiple papillae (Metcalfe 1960; Tateoka 1963; Renvoize 1985; Piperno and Pearsall 1998; Sánchez et al. 2003, 2006; Sánchez and Espinoza 2005). Luziola species also share features consistent with Oryzeae, such as oryzoid silica bodies, mesophyll with rosette cells [previously classified as arm cells by Ellis (1976)], and a complex midrib (Metcalfe 1960; Tateoka 1963; Pyrah 1967; Holm 1895).

Although rosette cells in the mesophyll and oryzoid silica bodies are two important features for recognizing Oryzeae, there are variations among the taxa within this tribe. Rosette cells are absent from the mesophyll in most oryzoids from Eurasia (Chikusichloa Koidz., Hygroryza Nees) and differences in the shape of the silica bodies is reported within Oryzeae species (Tateoka 1963). Despite the variation in shape, concerning the type of insertion of the silica bodies throughout the leaf blade, the presence of oryzoid-type silica bodies is a diagnostic feature only for oryzoids (Tateoka 1963; Ellis 1979; Prasad et al. 2005, 2011), and therefore, it is a useful characteristic for recognizing at least Oryzeae species.

The complex midrib as seen in Luziola and other genera of Oryzeae that includes at least two vascular bundles near to the abaxial surface (de Winter 1951; Tateoka 1963; Pyrah 1967; Renvoize 1985), seems to be a strong and distinctive feature between this tribe and Ehrharteae, which possesses only one vascular bundle in the midrib. Since the complex midrib is an important feature to delimit bamboo subtribes (BPG 2012), we need to mention that the complex midrib herein described is the only feature shared by all five species studied; therefore, potentially serving as a synapomorphy for Luziola or perhaps for the tribe Oryzeae—except for Hygroryza, which possesses a simple midrib. However, midrib structure in the other four species must be examined before any conclusions are possible. In addition, superposed mesophyll vascular bundles shared between L. spruceana and L. subintegra deserves more attention in future work with the anatomy of Oryzeae, since this feature seems unusual in Poaceae; to our knowledge, this is the first report for the grass family.

Another important feature also deserves attention. The vertically elongated epidermal cells surrounding unicellular trichomes, unique to L. fragilis, have not been previously described from any other grass species. We were not able to ascertain how these cells assume this characteristic or what function they may serve, thus we recommend an ontogenetic study to further explore this unique feature.

Fusoid cells are characteristic of Bambusoideae and the early diverging lineages, and thus are plesiomorphic within the Poaceae (GPWG 2001). Qualitative studies that report the presence or absence of these cells in the mesophyll of bamboos are common and taxonomically useful (e.g., Guala 1995; Oliveira et al. 2008; Leandro et al. 2016). Although the presence of fusoid cells has been a useful feature for species identification and delimitation, it is worth calling attention to the influence of light on the presence or absence of these cells. March and Clark (2011) compared the leaf blade anatomy in sun and shade leaves of bamboos, where leaves with and lacking fusoid cells were found even on the same plant, with the outermost leaves lacking fusoid cells. The influence of light and the function of fusoid cells are not yet clear, nonetheless, the presence of these cells in Luziola spruceana and L. subintegra is an important report toward understanding their occurrence and importance for Poaceae, since these species occur in open habitats.

Some xerophytic features, such as the epidermal furrows seen in L. fragilis and L. peruviana, would normally be expected in plants of more arid habitats (Oppenheimer 1960), rather than an aquatic plant. This feature may be a consequence of the establishment of these species in sites with variations of water availability, such as temporary ponds.

The results presented here show that the studied Luziola species possess consistent micromorphological and anatomical features of the leaf blade for the identification and delimitation of the taxa. Luziola spruceana and L. subintegra show the greatest morphoanatomical affinity, whereas L. fragilis shows the greatest number of exclusive features. Luziola peruviana shows only features common to all the other studied species. The results of the work by K. R. B. Leite are being prepared, and a combined analysis of the micromorphological and anatomical data for the entire genus is being planned, with the goals of characterizing the genus and understanding the evolution of anatomical and micromorphological features of the leaf blade within Luziola.

Taxonomic treatment

Key to the species of Luziola present in the Pantanal, Brazil, based on micromorphological and anatomical features

1a. Epidermis with vertically elongated cells surrounding pointed unicellular trichomes; midrib flat; leaf blade margins dimorphic … L. fragilis

1b. Epidermis without vertically elongated cells surrounding pointed unicellular trichomes; midrib with an abaxial keel; leaf blade margins structurally similar …………………………………………… 2

2a. Superposed vascular bundles in the mesophyll present; midrib composed of more than two vascular bundles; fusoid cells present …………………………………………… 3

2b. Superposed vascular bundles in the mesophyll absent; midrib composed of two vascular bundles; fusoid cells absent …………………………………………… 4

3a. Midrib with three vascular bundles superposed in a column; up to two air gaps …………………… L. spruceana

3b. Midrib with five vascular bundles superposed in a column; more than two air gaps …………………… L. subintegra

4a. Adaxial surface of the leaf blade with deep epidermal furrows; midrib with air gaps …………………… L. peruviana

4b. Adaxial surface of the leaf blade without epidermal furrows; midrib without air gaps …………………… L. bahiensis