The utility of Bambusoideae ( Poaceae , Poales ) leaf blade anatomy for identification and systematics

Bambusoideae is a diverse subfamily that includes herbaceous (Olyreae) and woody (Arundinarieae and Bambuseae) bamboos. Species within Bambusae are particularly difficult to identify due to their monocarpic lifecycle and the often long durations between mass flowering events; whereas the herbaceous bamboos are pluricarpic, but often are found with no reproductive structures. The leaf blade anatomy of 16 sympatric species of native Brazilian bamboos (Olyreae and Bambuseae) from the Atlantic Rainforest was studied in order to detect useful features for their identification. All the studied species share the following features: epidermis with a single stratum of cells; adaxial bulliform cells; mesophyll with arm cells, rosette cells, and fusoid cells; and collateral vascular bundles. Herbaceous bamboos share two features: papillae scattered on the abaxial surface and parallel-sided arrays of bulliform cells; whereas woody bamboos share: centrally organized papillae and fan-shaped arrays of bulliform cells. Also within the woody bamboos, intercostal fibers and a midrib with only one vascular bundle (simple midrib) characterize the subtribe Arthrostylidiinae; whereas a midrib with more than one vascular bundle (complex midrib) and a stomatal apparatus with two pappilae per subsidiary cell characterize the subtribe Chusqueinae. There are also diagnostic features for the sampled species, such as: papillae shape, and the outline and structure of the midrib. An identification key for all the studied species is provided based on the anatomical features.


Introduction
The cosmopolitan family Poaceae comprise about 11,000 species found mainly in grasslands and forest formations (Watson and Dallwitz, 1992 onwards;GPWG II, 2012).Twelve subfamilies are recognized within Poaceae (GPWG II, 2012;Soreng et al., 2015), among them Bambusoideae, a monophyletic group that currently includes 1,482 described species (Clark et al., 2015).Three Bambusoideae tribes are recognized, two of which are found in the Neotropics: Bambuseae, which comprise the woody bamboos; and Olyreae, the herbaceous bamboos (Kelchner, 2013;Clark et al., 2015).The Atlantic Rainforest is considered an important center of bamboo diversity (Judziewicz et al., 1999), and Brazil occupies a leading position based on number of species (298) and high endemism (172) (Carvalho et al., 2016).
Bambusoideae may be distinguished from other grass subfamilies by morphological, anatomical, and ecological characters.Monocarpic perennial lifecycle, lignified culms, branching nodes, pseudopetiolate leaves, and an outer ligule are characters worth mentioning for the woody bamboos (GPWG, 2001;BPG, 2012); whereas herbaceous bamboos are pluricarpic, usually unbranched, with quite weak culms and an inner ligule (Judziewicz et al., 1999).Together with, the strongly and asymmetrically invaginated arm cells as seen in cross section are highly important for the recognition of Bambusoideae species (GPWG, 2001), and also represents one of the main synapomorphies for this group (Zhang and Clark, 2000;BPG, 2012).
In general, the Poaceae taxonomy is mainly based on reproductive characters, such as the shape and structure of spikelets and inflorescence types (Longhi-Wagner, 2012).This is true more particularly for the herbaceous species, which generally bloom many times in their life cycle.In contrast, the woody bamboos bloom only once during a life cycle (Janzen, 1976;Filgueiras, 1988), and sometimes even herbaceous species are found with no reproductive structures.For this reason, searching for vegetative characters in addition to the reproductive ones is highly important to aid in species identification, and anatomical features often have provided useful findings (e.g.Brandis, 1907;Prat, 1936;Brown, 1958;Metcalfe, 1960;Calderón and Soderstrom, 1973;Renvoize, 1987;Vieira et al., 2002;Guglieri et al., 2008;Oliveira et al., 2008;Pelegrin et al., 2009;Jesus Junior et al., 2012;Viana et al., 2013a, b;Leandro et al., 2016;Aliscioni et al., 2016).
Considering that mostly bamboo plants have unique life cycles, but also the importance of the leaf blade anatomy for the taxonomy of grasses in general, we studied 16 sympatric species of native bamboos from the Atlantic Rainforest.We examined the leaf blade anatomy of three species of herbaceous bamboos and 13 species of woody bamboos in order to provide useful features for their identification.

Sampling area
The study was carried out with 16 native species sampled at Parque Estadual das Fontes do  [Ipiranga State Park], a fragment of Atlantic Rainforest located in the State of São Paulo, Brazil.We have analysed three specimens per species, but only one voucher per specimens was included in the herbarium of the Instituto de Botânica (SP) (Table 1).The choice of taxa was based on a floristic study of the area that indicated the necessity of providing additional data in order to aid in species identification and conservation (Shirasuna and Filgueiras, 2013).Olyra loretensis Mez was not included in this study due to its uncertain occurrence in the PEFI (see Shirasuna and Filgueiras, 2013 for details about each species).

Anatomical analysis
For the woody bamboos, mature leaf blades were taken from the branches at the mid-culm, whereas for the herbaceous bamboos mature leaves were taken from the third node from the base.Fresh plant material was fixed in FAA 50 (Johansen, 1940) and later stored in 70% ethanol.Found on leaves of Arthrostylidiinae species, the green stripe was excluded from this work due to its anatomical peculiarities in relation to the remainder of the leaf blade (Judziewicz et al., 1999).
Samples from the middle portion of the leaf blade were embedded in polyethylene glycol 1500 solution (adapted from Richter, 1985) and cross-sectioned with a rotary microtome.Sections were cleared in sodium hypochlorite 50%, washed in distilled water, stained with Astra blue and Safranin (Bukatsh, 1972), and finally mounted on semi-permanent slides with glycerol.Also, a maceration technique was performed by the Jeffrey's method (Johansen, 1940) in order to describe the epidermal features.
Descriptions were primarily based on Ellis (1976Ellis ( , 1979)), and optical images were obtained on a Leica DM4000B microscope using the software Leica Application Suite LASV4.0.
A scattered distribution of papillae is observed in the herbaceous bamboos (e.g. Figure 1D; Table 2), whereas the organization in the woody bamboos is often in a single central row (e.g.Figures 1G, I), but may be variable in some intercostal cells (1-2 rows) (e.g. Figure 1H).
Trichomes mainly occur on the abaxial surface and they may be of three types: (i) prickle hairs (short and silicified, microscopic unicellular) (Figures 1C, F 1E, F).Prickle hairs of most of the species develop an enlarged base, usually as seen in Chusquea capituliflora var.pubescens (Figure 1F), but in M. neesii this base is more pronounced (Figure 1C).Macrohairs occur on the abaxial surface of C. capituliflora var.pubescens (Figure 1F) and Chusquea meyeriana Rupr.ex Doell (Figure 1I -scars).Bicellular microhairs often consist of cells of about the same size (e.g. Figure 1M), except for C. capituliflora var.pubescens, in which the apical cell is reduced (Figure 1L).Microhairs often occur on the abaxial surface in the woody species and Parodiolyra micrantha (Kunth) Davidse & Zuloaga.
Stomata are paracytic and occur on the abaxial surface of all the studied species, but also on the adaxial surface in A. aristulata and Olyra humilis Nees (Figure 1B; Table 2).Stomatal apparatus comprise triangular subsidiary cells (Figures 1B, D, H, K) or semi-circular (cupuliform) cells (Figures 1A, E, G).In species of Chusquea the stomatal apparatus bears two papillae per subsidiary cell as seen in Chusquea capituliflora var.pubescens (Figure 1E detail inset; Table 2).

Cross section
The epidermis consists of a single stratum of cells with slightly thickened outer walls (Figures 2A-Q).Epidermal cells are visually about the same size (Figures 2E, G, J), but may be larger on the adaxial side (Figures 2F, H, M)excluding the bulliform cells.Bulliform cells occur as part of the adaxial epidermis (Figures 2D-M) and form a fanshaped array in the woody bamboos, (e.g.Figures 2G, J, K; Table 2); whereas the herbaceous bamboos share a parallelsided array of bulliform cells (e.g.Figures 2E, I; Table 2).
The mesophyll comprise arm cells, fusoid cells, rosette cells, fibers, and vascular bundles.Asymmetrically invaginated arm cells are parallel to the epidermis (Figures 2E-M), including the midrib portion (Figures 2A-D).Herbaceous species (Figures 2E, I) and Chusquea bambusoides (Figure 2H) develop arm cells with invaginations only from the abaxial side, whereas the other species develop invaginations from both sides (Figures 2F, G, J-M).The number of rosette cells between each fusoid cell is often variable (one to four) within the same sample/specimen (Figures 2E-M).Fusoid cells occur adjacent to the vascular bundles and arm cells (Figures 2E-M); and their outline may be short and wide, as seen in A. aristulata (Figure 2G), or long and narrow, as in M. neesii (Figure 2F).Intercostal fibers located adjacent to the bulliform cells (sometimes also opposite) occur just   2A-M): the outer one is parenchymatic and may be interrupted by fibers from both sides, as seen in Merostachys speciosa Spreng.(Figure 2M) or only from the abaxial side, as in Merostachys skvortzovii Send.(Figure 2L); and the inner one is pericyclic (mestome) with thick-walled cells (Figures 2A-M).First and third order vascular bundles are observed in all the studied species (Figures 2F-M).
In most of the studied species the midrib is flat (e.g. Figure 2D), but it is abaxially projected in species of Chusquea (Figures 2A, B) and adaxially projected in Padoriolyra micrantha (Figure 2C; Table 2).The midrib comprise one first order vascular bundle (Figures 2C, D), except among species of Chusquea, in which the midrib includes minor vascular bundles adjacent to the central one (Figures 2A, B; Table 2).
With regard to the margin, the leaf blade may be acute (Figure 2N-P) or obtuse (Figures 2Q); always with thickwalled epidermal cells and fibers immediately subjacent to the epidermis (e.g.Figures 2N-Q).

Taxonomic treatment
The main anatomical features are summarized in Table 2.These data in tabular form are available upon request from the first author.
Identification key to the native Bambusoideae species from PEFI, SP, based on the leaf blade anatomical data (surface view and cross section):

Discussion
Our anatomical study demonstrates that papillae scattered on the abaxial surface and parallel-sided arrays of bulliform cells are exclusive features among the herbaceous bamboos sampled; whereas centrally organized papillae and fan-shaped arrays of bulliform cells are exclusive features among the woody bamboos sampled.
Within the herbaceous bamboos sampled, the midrib outline and amphistomatic leaves may distinguish Parodiolyra Soderstr.& Zuloaga from Olyra L. Although this may be true, it is not clear if these features are consistent among all Brazilian species of Olyra (20) and Parodiolyra (four) (Oliveira and Filgueiras, 2016a, b).Comparatively, within the woody bamboos sampled, intercostal fibers and a midrib with only one vascular bundle (simple midrib) characterize the subtribe Arthrostylidiinae; whereas a stomata apparatus bearing two papillae per subsidiary cell and a midrib with more than one vascular bundle (complex midrib) characterize the subtribe Chusqueinae.The presence of two papillae per subsidiary cell herein supports the assumption of this feature as a synapomorphy for Chusquea (Fisher et al., 2009(Fisher et al., , 2014)), although there are not enough studies on micromorphology and anatomy to clarify its value.Currently, the set of features herein observed for Arthrostylidiinae and Chusqueinae is common among all species known within each subtribe and extremely applicable for recognizing these groups (BPG, 2012;Clark et al., 2015).
The comparative anatomical analysis herein performed demonstrates that the variation in the distribution of papillae is useful for delimiting tribes.There are some reports showing the importance of this feature in bamboo systematics (e.g.Soderstrom and Ellis, 1987;Paisooksantivatana and Pohl, 1992;Yang et al., 2008;Gomes and Neves, 2009;Mota, 2013), but also for other closely related groups (e.g.Pelegrin et al., 2009).Our study is not able to define the value of this feature to the systematics of Olyreae and Bambuseae, therefore a detailed work to evaluate both distribution and type of papillae within different groups would be informative.
Our study also indicates that some features may be considered diagnostic at the species level.Among them, the stomata on the adaxial surface in Aulonemia aristulata must be mentioned, since their occurrence is considered as rare for Aulonemia (Arthrostylidiinae) (Viana et al., 2013a), but usually typical for species within the subtribe Guaduineae (Soderstrom and Ellis, 1987).Adaxial stomata were also recently observed in other species within Aulonemia (Viana, 2010;Viana et al., 2001), and thus it reinforces the anatomical affinity between the subtribes Arthrostylidiinae and Guaduineae (Bambuseae) (Soderstrom and Ellis, 1987;Zhang and Clark, 2000;Ruiz-Sanchéz et al., 2008), as well as the necessity of a broad anatomical study in order to elucidate the systematic value of this feature for Bambuseae.
It is important to highlight that the size and shape of bulliform cells may be influenced by environmental factors (Shields, 1951), but the structural variation herein observed deserves more attention in order to verify its constancy among bamboo groups.Also, the fusoid cell is another feature that requires additional attention since its environmentally influenced morpho-anatomical variations (March and Clark, 2011;T. D. Leandro, unpubl. data).
In the present study, we consider the structure of bulliform cells and the outline of fusoid cells as relevant features for delimiting species given that all specimens were sampled under the same environmental conditions.

Conclusion
Although most of the information herein provided is not a novelty for Bambusoideae, our results reinforce the importance of leaf blade anatomy studies for grass systematics, specially when we consider the great number of questions that are still unclear.The inclusion of anatomical data as a routine on bamboo studies may be really useful for identifying diagnostic features and additional synapomorphies, in which certainly will aid in species circumscription.

Table 1 .
Specimens used in this study, with classification and voucher information provided.

Table 2 .
Summary of leaf blade features useful for delimiting the tribes and subtribes, and also for recognizing the species.