ORIGINAL ARTICLE

The contribution of foliar micromorphology and anatomy
to the circumscription of species within the Chusquea ramosissima
informal group (Poaceae, Bambusoideae, Bambuseae)

Thales D. Leandro1 • Vera L. Scatena1 • Lynn G. Clark2

Received: 12 August 2016 / Accepted: 15 March 2017 / Published online: 7 April 2017

� Springer-Verlag Wien 2017

Abstract Chusquea is a diverse but monophyletic genus of

Neotropical woody bamboos from primarily montane for-

ests that comprises four well-supported lineages: subg.

Magnifoliae, subg. Platonia, subg. Rettbergia, and the

Euchusquea clade (comprising subg. Swallenochloa and

subg. Chusquea). However, the relationships among clades

or taxa within the Euchusquea clade inferred from molec-

ular data are mostly not congruent with those inferred from

morphological evidence, consequently limiting our ability

to understand species relationships. In this study we gen-

erated foliar micromorphological and anatomical data for

the Chusquea ramosissima informal group (Chusquea

ramosissima, C. tenella, and C. tenuiglumis), and for the

putative new species from Bolivia in this group, in order to

test the value of these types of data for defining species and

to seek potential synapomorphies for this group. Our

results demonstrate that epidermal features, mainly with

regard to the stomatal apparatus, proved to be more valu-

able in distinguishing species than anatomical characters.

The presence of horizontally elongated silica cells over the

veins and adaxial arm cells with invaginations from the

abaxial side was shared by all the studied species but is not

unique to this group. The type of trichomes, shape of silica

bodies, type of arm cells, and midrib structure may be

useful to lesser degree. All four species exhibited

intraspecific variation in development of the papillae on the

long cells. Support for the recognition of the new species

from Bolivia is provided by micromorphological charac-

ters. An identification key based on leaf blade features is

provided for the four studied species.

Keywords Chusqueinae � Leaf � Monocotyledons �
Poales � Woody bamboos

Introduction

Bambuseae comprise one of three lineages within Bam-

busoideae and include at least 812 species in 66 genera

(Clark et al. 2015). This tribe is mostly diversified in

tropical forests, both lowland and montane, and mostly in

relatively humid habitats (Judziewicz et al. 1999; Clark

et al. 2015). Two major lineages are currently recognized

for Bambuseae based on molecular data (Sungkaew et al.

2009; Kelchner and BPG 2013): Paleotropical woody

bamboos (seven subtribes, 407? species), and Neotropical

woody bamboos (three subtribes, 405? species) (BPG

2012; Clark et al. 2015; Wong et al. 2016). The phyloge-

netic relationship of the three subtribes within the

Neotropical woody bamboos [(Arthrostylidi-

inae ? Guaduinae) ? Chusqueinae] has good support

(Kelchner and BPG 2013; Wysocki et al. 2015), although

no defining characters have been identified for the

Neotropical clade (Clark et al. 2015). Sister to each other,

Arthrostylidiinae and Guaduinae are each well supported

as monophyletic based on morphological and molecular

data (Ruiz-Sanchez 2011; Tyrrell et al. 2012; Kelchner and

BPG 2013), while the monophyly of Chusqueinae, which

includes only the genus Chusquea Kunth, is based pri-

marily on molecular evidence although all members share

Handling editor: Mike Thiv.

& Thales D. Leandro

thaleshdias@gmail.com

1 Departamento de Botânica, Universidade Estadual Paulista,

Av. 24A, 1515, Rio Claro, SP 13506-900, Brazil

2 Department of Ecology, Evolution, and Organismal Biology,

Iowa State University, Ames, IA 50011-1020, USA

123

Plant Syst Evol (2017) 303:745–756

DOI 10.1007/s00606-017-1404-0

http://orcid.org/0000-0002-5800-5432
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the presence of two papillae per subsidiary cell and one-

flowered spikelets with four glumes and no rachilla

extension (Fisher et al. 2009, 2014; Kelchner and BPG

2013).

Chusquea is the most diverse genus of Neotropical

woody bamboos, comprising more or less 175 described

species primarily from montane forests with some inhab-

iting high elevation grassland formations (Judziewicz et al.

1999; Fisher et al. 2014). Plastid sequence data support the

existence of four clades within Chusquea (Fisher et al.

2009, 2014): (1) subg. Magnifoliae; (2) subg. Platonia; (3)

subg. Rettbergia; and (4) the major Euchusquea clade,

comprising subg. Swallenochloa and subg. Chusquea, and

including about 75% of the species diversity of the genus.

However, inferring relationships within the Euchusquea

clade with confidence remain a challenge because its

morphological-based subgeneric and sectional classifica-

tion (Clark 1989; Fisher et al. 2009, 2014) is largely

incongruent with the molecular topology. On the other

hand, the Chusquea ramosissima informal group, which is

distinguished by a unique combination of morphological

characters within the Euchusquea clade, also was supported

in the molecular phylogeny (Fisher et al. 2009, 2014).

The C. ramosissima informal group, defined by the

presence of culm leaves with pseudopetiolate, deciduous

blades that usually remain green and racemose or weakly

paniculate synflorescences, consists of C. ramosissima

Lindman, C. tenella Nees, and C. tenuiglumis Döll (Fisher

et al. 2009, 2014). These species are distributed in

Argentina, Bolivia, Brazil, Paraguay, and Uruguay, and

occupy Atlantic or montane forests from sea level to

1200 m in elevation (Judziewicz et al. 1999). Previously,

these three species were included within subg. Rettbergia

(Judziewicz et al. 1999); however, Fisher et al.

(2009, 2014) and Mota (2013) excluded them from subg.

Rettbergia and placed them within subg. Chusquea, based

on their infravaginal branching and the more or less con-

stellate arrangement of subsidiary buds with respect to the

central bud (Clark 2004), as well as the unambiguous

resolution of C. ramosissima and C. tenella within the

Euchusquea clade based on plastid data.

Woody bamboos grow vegetatively for long periods,

and then their life cycle usually ends after a single gre-

garious flowering episode (Seifriz 1950; Janzen 1976;

Judziewicz et al. 1999; Ruiz-Sanchez et al. 2017). For this

reason, vegetative morphological data from rhizomes,

culms, branches, and leaves remain extremely important

for bamboo studies. Anatomical and micromorphological

surveys in addition to morphological ones have established

the importance of these types of features in providing the

basis for a more natural classification within the grass

family (e.g., Guglieri et al. 2008; Oliveira et al. 2008;

Gomes and Neves 2009; Pelegrin et al. 2009; Aliscioni

et al. 2016; Leandro et al. 2016a, b). Although diagnosable

macro-morphological characters of Chusquea and its sub-

genera have been identified (Fisher et al. 2009, 2014), there

is a lack of knowledge with respect to the value of leaf

blade anatomical and micromorphological features across

the genus, even considering the amount of data available in

the literature (e.g., Clark 1986, 1990; Clark et al. 1989;

Montti et al. 2008, 2009; Guerreiro et al. 2013; Lizarazu

2013).

Thus, considering the incongruence between the

chloroplast phylogeny versus the morphology-based clas-

sification of the Euchusquea clade, and the need for

anatomical and micromorphological studies in the genus,

the main objective of the current work was to examine leaf

blade micromorphology and anatomy and test their value

for circumscription across species within the morphologi-

cally based C. ramosissima informal group (Fisher et al.

2009, 2014). To that end, C. ramosissima, C. tenella, and

C. tenuiglumis were studied. We also included a putative

undescribed species of Chusquea from Bolivia that belongs

to this group and is morphologically similar to C.

ramosissma.

Materials and methods

Taxon sampling

The following three species comprising the C. ramosissima

informal group (Fisher et al. 2009, 2014) were studied: C.

ramosissima, C. tenella, and C. tenuiglumis. Chusquea

ramosissima is widely distributed in Argentina, Bolivia,

Brazil, Paraguay, and Uruguay, C. tenella occurs in

Argentina, Brazil and Uruguay, and C. tenuiglumis is

endemic to Brazil. Depending on available material, we

balanced sampling to represent the geographic ranges of

these species as well multiple populations in relative

proximity (Table 1), but we did not include any material

from Uruguay as the only available specimens were not in

good condition. We also included three samples of a

putative new species from Bolivia referred to as Chusquea

sp. nov. (Table 1). The observation of specimens using

scanning electron microscopy (SEM) or light microscopy

(LM) is indicated in Table 2.

Material for both micromorphology and anatomy was

taken from fully expanded leaf blades in the middle of the

branch complement, from either sterile or fertile herbarium

specimens. For both C. ramosissima and C. tenella, a

majority of available specimens were fertile, as bamboos

tend to be collected by non-specialists only when in flower,

but for each species, two or more sterile collections were

also available and sampled. Available specimens for C.

tenuiglumis and Chusquea sp. nov. were all sterile; the only

746 T. D. Leandro et al.

123



Table 1 Voucher information for the four species of the Chusquea ramosissima informal group used in this study

Chusquea ramosissima Lindm.

BRAZIL. Paraná: Mun. Araucaria, margins of the floodplain of the Rio Iguaçu, near the bridge crossing of BR-476, Curitiba-Lapa, 880 m a. s.

l., 3 Mar 1991, Clark and Windisch 849 (ISC); Mun. Santa Helena Porto Verde, 12 Dec 1977 (fl), Hatschbach 40533 (US); Mun. Cerro

Azul, Cab. Rib. do Tigre, 24 Jan 1980 (fl), Hatschbach 42746 (US). Rio Grande do Sul: Boa Vista, Salvador do Sul, 200 m a. s. l., 22 Apr

1969, Klein 8307 (US). Rio de Janeiro: Mun. de Petŕopolis, Vale de Bom Sucesso, ‘‘Caixa de Agua’’, 720 m a. s. l., 6 May 1972, Soderstrom

and Sucre 1986 (US). Santa Catarina: Santo Antônio, Itapiranga, 400 m a. s. l., 20 Jan 1974, Klein and Klein 11041 (US). PARAGUAY.

Dep. Guairá: Colonia Independencia, 1 Apr 1950, Anderson 1188 (US). Dep. Itapúa: Obligado, El Tirol, 19.5 km by road NNE of

Encarnación, 6 Nov 1976, Foster 76-35 (US)

Chusquea tenella Nees

ARGENTINA. Misiones: Morro Sapucaia, 29 Jan 1948 (fl), Palacios-Cuezzo 474 (US). BRAZIL. Paraná: Campo Morão, 28 Mar-2 Apr 1946,

Swallen 9004 (US). Rio Grande do Sul: Vila Manresa p. P. Alegre, 15 May 1950 (fl), Rambo 47076 (US). Santa Catarina: Morro do Rio

Vermelho, 250 m a. s. l., 27 Jun 1968 (fl), Klein and Bresolin 7766 (US); Palmas, Governador Celso Ramos, 100 m a. s. l., 19 May 1971 (fl),

Klein and Bresolin 9477 (US). São Paulo: BR-116, ca. Km 515, about 38 km S of Jacupiranga, Braço Feio, Serra do Azeite, 400 m a. s. l., 4

Mar 1990, Clark and Windisch 725 (ISC)

Chusquea tenuiglumis Döll in Martius

BRAZIL. Minas Gerais: Mun. Viçosa, municipal road from Viçosa to Araponga, 730 m a. s. l., 25 Feb 1990, Clark and Morel 706 (ISC); Rio

de Janeiro: Estrada Rio de Janeiro-Grumarı́, 100 m a. s. l., 23 Mar 1972, Soderstrom and Sucre 1918 (US); Santa Catarina: Mun.

Petrolândia, 3 km before Petrolândia, 530 m a. s. l., 5 Mar 1991, Clark and Windisch 864 (ISC)

Chusquea sp. nov.

BOLIVIA. Dep. La Paz, Prov. Franz Tamayo: Parque Nacional Madidi, camino de Apolo-Azariamas, Arroyo Pintata, a 495 m del

campamento en dirección SE, aprox. 20 minutos saliendo del camino, 1011 m a. s. l., 19 Feb 2003, Cayola et al. 6 (ISC); Parque

Nacional Madidi, Chaquisapa, cerca al rio Mojos, 921 m a. s. l., 9 Jul 2005, Fuentes et al. 9662 (ISC); Parque Nacional y Area

Natural de Manejo Integrado Madidi, norte de Apolo, 940–1170 m a. s. l., 7 Mar 2005, Uzquiano et al. 10 (MO)

ISC Ada Hayden Herbarium at Iowa State University; MO Herbarium at the Missouri Botanical Garden; US U.S. National Herbarium at the

Smithsonian Institution

Table 2 List of Chusquea

species, vouchers and their use

in this study

Taxa Voucher Analysis performed

SEM LM

Chusquea ramosissima Anderson 1188 X

C. ramosissima Hatschbach 40533 X X

C. ramosissima Foster 76-35 X

C. ramosissima Hatschbach 42746 X X

C. ramosissima Clark and Windisch 849 X

C. ramosissima Soderstrom and Sucre 1986 X

C. ramosissima Klein 8307 X

C. ramosissima Klein and Klein 11041 X

Chusquea tenella Klein and Bresolin 9477 X

C. tenella Klein and Bresolin 7766 X X

C. tenella Clark and Windisch 725 X

C. tenella Swallen 9004 X

C. tenella Palacios-Cuezzo 474 X

C. tenella Rambo 47076 X

Chusquea tenuiglumis Clark and Morel 706 X X

C. tenuiglumis Clark and Windisch 864 X X

C. tenuiglumis Soderstrom and Sucre 1918 X

Chusquea sp. nov. Fuentes et al. 9662 X X

Chusquea sp. nov. Cayola et al. 6 X X

Chusquea sp. nov. Uzquiano et al. 10 X X

LM light microscopy, SEM scanning electron microscopy

Foliar micromorphology and anatomy of the Chusquea ramosissima informal group 747

123



flowering specimen of C. tenuiglumis is the type with

limited leaf material, and thus we did not sample it.

Micromorphology (SEM)

Two pieces of approximately 0.5 cm2 of the middle portion

of a fully expanded branch leaf were excised and attached

to a cylindrical sample holder (stub) such that both adaxial

and abaxial surfaces were exposed, and coated with a thin

sample of gold (Denton vacuum Desk III). At least two

specimens per species were submerged in xylene for

approximately 10 min, eliminating the epicuticular wax to

provide a better view of micromorphological features

(Dávila and Clark 1990). Observation and images of the

surface view were obtained with the aid of a scanning

electron microscope (JEOL JSM-5800LV) in the Micro-

scopy and Nano-Imaging Facility (MNIF) at Iowa State

University (ISU).

Anatomy (LM)

The middle portion of fully expanded branch leaves was

excised and then immersed in polyethylene glycol 1.500

solution and kept in an incubator at 60 �C for fifteen days

(adapted from Richter 1985). Cross sections were made

using a Spencer 820 rotary microtome, and then they were

cleared in 50% sodium hypochlorite, rinsed in distilled

water, and stained in epoxy tissue stain (Spurlock et al.

1966). Semi-permanent slides were mounted in 50%

glycerin and analyzed under a light microscope (Olympus

BX-40).

Leaf clearings were performed in order to describe the

structures in surface view. For this purpose, pieces of

approximately 0.5 cm2 from the middle portion of a fully

expanded branch leaf were removed. 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 1%

safranin and 0.5% fast green in ethanol (adapted from

Johansen 1940). Stained samples were treated with ethyl

alcohol (95 and 100%), washed with xylene and then with

xylene and Permount (1:1). Permanent slides were moun-

ted with Permount.

Images and terminology

For both sections and clearings, photomicrographs were

obtained with the aid of a Zeiss Axio Observer microscope

in the MNIF at ISU using ZEN 2.0 blue software, and

applying different light contrast regimes. The terminology

for the leaf blade in cross section and in surface view was

primarily followed Ellis (1976, 1979), but updates in leaf

blade terminology were considered as appropriate. The

widely employed term ‘‘fusoid cells’’ was used as a proxy

for the usually conspicuous intercellular spaces (often

including cell wall remnants) produced by fusoid cell

collapse during development as seen in cross section (Vega

et al. 2016).

Results

The following data in tabular form are available upon

request from the first author. A summary of the most

informative micromorphological and anatomical features

for each species is provided in Table 3.

Epidermal surfaces (SEM and clearings)

Data for epidermal cells as seen with scanning electron

microscopy (Figs. 1, 2a–c) and with light microscopy of

clearings (Fig. 2d–m) are merged in order to provide a

better understanding.

The epidermis consists of alternating long and short

cells (Figs. 1a–i, 2a–f, i–l) covered with a layer of epicu-

ticular wax (e.g., Fig. 1a). In general, long cells are tabular

shaped with thin, undulating anticlinal walls (Figs. 1c, i,

2i). Intercostal long cells on both leaf blade surfaces often

bear dome-shaped papillae that are often organized in one

or two central rows (Figs. 1a, c–f, 2a–c, l), but only scat-

tered papillae may be observed on the abaxial long cells of

C. ramosissima (Fig. 1b) and the adaxial ones of C.

tenuiglumis (Fig. 1g). Papillae are usually absent or poorly

developed in the interstomatal band of the intercostal zone

in C. ramosissima (Fig. 1b) and C. tenuiglumis (Fig. 1h, i),

but often are well developed in C. tenella (Fig. 1e, f) and

Chusquea sp. nov. (Fig. 2b).

Short cells may be developed as silica cells or cork cells

(Fig. 2g, l). Silica cells occur in the costal zone on both

surfaces and they are associated with tabular short cells

(e.g., Fig. 2k), but they also occur scattered on the adaxial

surface (sometimes also abaxially) in the intercostal zone

(e.g., Figs. 1d, e, 2a, f). In the costal zone (over the veins),

silica cells are horizontally oriented (e.g., Fig. 2d, e, j, k),

but in the interstomatal band of the intercostal zone, silica

cells are always vertically oriented (e.g., Fig. 2f, l, m).

With regard to shape, silica cells are mainly saddle-shaped

(e.g., Fig. 2j), but dumbbell-shaped (Fig. 3d, e, k) and

irregular-crenate cells (Fig. 2d) can be observed in the

same specimen/sample. Also, rarely, intercostal long cells

might be silicified forming elongated tabular crenate silica

cells (Fig. 2i). Cork cells are tall and narrow and occur as

silico-suberose pairs mostly in the intercostal zone

(Fig. 2g, l).

748 T. D. Leandro et al.

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Stomata are always observed on the abaxial epidermis in

rows (Figs. 1b, c, e, f, h, i, 2b, c, e, f, m), but they occur

scattered on the adaxial epidermis in C. tenuiglumis

(Fig. 1g), and Chusquea sp. nov. (Fig. 3c, arrowhead). On

the abaxial epidermis, the number of rows of stomata is

variable (one to three) among species (e.g., Fig. 1b, c, f, h,

i), and usually there is only one interstomatal long cell

separating successive stomata in a row (e.g., Fig. 1c, i).

The stomatal apparatus is paracytic (e.g., Fig. 2m) with

subsidiary cells that are low dome shaped (Fig. 2f) or low

triangular shaped (Fig. 2m) and bearing two papillae each,

the papillae branched (Fig. 1c, i) or not (simple) (Figs. 1f,

2c). Branched papillae completely overarch the guard cells

and form a chamber above the pore (Fig. 1c, i). In C.

tenella and Chusquea sp. nov., the simple papillae arch

over and meet across the guard cells, but do not form a

chamber (Figs. 1f, 2c).

Trichomes can be of five types: (1) silicified and pointed

unicellular with the base forming an integral part of the

epidermis (prickle hair); (2) silicified unicellular with a cell

that emerges straight out of the epidermis (microhair); (3)

non-silicified unicellular (macrohair); (4) non-silicified

bicellular with a basal and a distal cell (microhair); (5) non-

silicified bicellular with many specialized epidermal cells

adjacent to the hair base (microhair). Prickle hairs occur on

both surfaces in C. tenella (Fig. 1d, e) and Chusquea sp.

nov. (Fig. 2b, c). Non-silicified macrohairs occur on both

surfaces of Chusquea sp. nov. (Fig. 2a, b) and may be

observed on the abaxial surface of C. ramosissima and C.

tenella (Fig. 2e) Silicified unicellular microhairs occur

often on the abaxial surface (rarely adaxially) in C. tenella

and Chusquea sp. nov. (Fig. 2a, h), whereas bicellular

microhairs occur on the abaxial surface of all the studied

species (e.g., Figs. 1b, e, h, 2b).

Cross section

The outline of the leaf blade as seen in cross section is

gently undulated (e.g., Fig. 3a, b). The epidermis consists

of a single layer of cells with slightly thickened outer

periclinal walls in most of the species (e.g., Fig. 3a, b, d, e).

Epidermal cells are all about the same size except for the

presence of fan-shaped arrays of bulliform cells in between

the bundles as part of the adaxial epidermis (e.g., Fig. 3a,

b). The number of bulliform cells in each array is variable

among samples within the same species.

The mesophyll comprises asymmetrically invaginated

arm cells, fusoid cells, and rosette cells (e.g., Fig. 3a–c).

Arm cells are organized in two layers (e.g., Fig. 3a–c),

and invaginations of these cells are from the abaxial side

(e.g., Fig. 3a–c). Just one layer of arm cells is observed

adjacent to the abaxial surface, in which the invagina-

tions are relatively shallow and often occur just from the

adaxial side (e.g., Fig. 3a, b). Surrounded by arm cells

and adjacent to the vascular bundles, the fusoid cells are

highly variable in shape and size among and within

Table 3 Summary of the main

foliar micromorphological and

anatomical features of the

Chusquea ramosissima informal

group: 1. Chusquea

ramosissima; 2. C. tenella; 3. C.

tenuiglumis; 4. Chusquea sp.

nov.

Features Taxa

1 2 3 4

Surface view

Epidermis

Silica bodies (costal) horizontally oriented ? ? ? ?

Silica bodies (costal) vertically oriented - - - -

Subsidiary cells bearing two branched papillae ? - ? -

Subsidiary cells bearing two simple papillae - ? - ?

Subsidiary cells low dome shaped - - - ?

Subsidiary cells low triangular shaped ? ? ? -

Cross section

Mesophyll

Arm cells with invaginations from the abaxial side ? ? ? ?

Arm cells with invaginations from both sides - - - -

Midrib

Composed of two vascular bundles ? - ? ?

Composed of three vascular bundles - ? - -

Margins

Dimorphic ? - - –

Structurally similar - ? ? ?

? Presence, - absence

Foliar micromorphology and anatomy of the Chusquea ramosissima informal group 749

123



Fig. 1 Micromorphology from SEM of Chusquea ramosissima

informal group species. Adaxial surface (a, d, g), abaxial surface

(b, c, e, f, h, i). a C. ramosissima (Hatschbach 40533); b C.

ramosissima (Hatschbach 42746); c C. ramosissima (Soderstrom and

Sucre 1986); d C. tenella (Klein and Bresolin 7766, inset Swallen

9004); e C. tenella (Swallen 9004); f C. tenella (Palacios-Cuezzo 474,

inset Swallen 9004); g C. tenuiglumis (Clark and Windisch 864). h C.

tenuiglumis (Clark and Morel 706, inset Hatschbach 42746); i C.
tenuiglumis (Clark and Morel 706). ilc intercostal long cell, ma

macrohair, mi microhair, pa papilla, pr prickle, sc silica cell, st

stomatal apparatus. Scale bars 60 lm (a, b, d, e, g, h), 13 lm (c, f, i),
6.5 lm (insets)

750 T. D. Leandro et al.

123



Fig. 2 Micromorphology (SEM) and anatomy (light microscopy) of

Chusquea ramosissima informal group species in surface view.

Adaxial surface (a, d, f, g, i, l), abaxial (b, c, e, h, j, k, m).

a Chusquea sp. nov. (Fuentes et al. 9662, inset Uzquiano et al. 10);

b Chusquea sp. nov. (Cayola et al. 6); c Chusquea sp. nov. (Fuentes

et al. 9662); d Chusquea sp. nov. (Cayola et al. 6); e C. ramosissima
(Clark and Windisch 849); f Chusquea sp. nov. (Uzquiano et al. 10);

g Chusquea sp. nov. (Cayola et al. 6); h Chusquea sp. nov. (Fuentes

et al. 9662) showing a silicified unicellular microhair; i C. tenella
(Clark and Windisch 725); j Chusquea sp. nov. (Cayola et al. 6); k C.

ramosissima (Clark and Windisch 849); l Chusquea sp. nov. (Cayola

et al. 6); m C. ramosissima (Clark and Windisch 849) showing

papillae (arrowheads). cc cork cell, csc costal short cell, ds dumbbell

shaped silica cell, gc guard cells, hsc horizontally oriented silica cell,

ilc intercostal long cell, isc irregular-crenate silica cell, ma macrohair,

mi microhair, pa papilla, pr prickle, sc silica cell, smi silicified

unicellular microhair, ss saddle shaped silica cell, st stomatal

apparatus, su subsidiary cell, tcs tabular crenate silica cell, vn vein.

Scale bars 5 lm (g–m), 6.5 lm (inset), 10 lm (f), 20 lm (d, e),
60 lm (a, b), 13 lm (c)

Foliar micromorphology and anatomy of the Chusquea ramosissima informal group 751

123



species/specimens (e.g., Fig. 3a–e). These cells occur

along the entire lamina, except at the margins. Rosette

cells occur between fusoid cells (e.g., Fig. 3a, b), with

the number of cells extremely variable in each group

along the leaf blade.

With regard to the vascular tissue, xylem and phloem

are collateral and surrounded by a double sheath, the inner

mestome sheath and the outer parenchymatic sheath (e.g.,

Fig. 3a, b). Vascular bundles comprise two types: (1) first

order with distinguishable metaxylem and phloem (e.g.,

Fig. 3a, b); and (2) third order, small vascular bundles with

usually a few lignified tracheary elements and a small patch

of phloem (e.g., Fig. 3a, b). Girders occur in both types of

vascular bundles, but only the parenchymatic sheath from

the first-order vascular bundles is abaxially interrupted by

this arrangement of fibers (e.g., Fig. 3b). The midrib is

complex and is composed of one large abaxial and one

smaller adaxial opposing vascular bundle (e.g., C. tenuig-

lumis, Fig. 3d) or one large abaxial and two smaller

opposing bundles (C. tenella, Fig. 3e), all sharing the same

bundle sheaths.

Leaf blade margins are mainly acute, but one acute and

one obtuse margin (dimorphic) may be observed within the

same leaf blade of C. ramosissima (Table 3). Also, the

amount of sclerenchyma cells in this region is variable

among species and even within the same sample.

Discussion

Micromorphological and anatomical data

At least some long cells with papillae; fan-shaped arrays of

bulliform cells; a mesophyll with some strongly invagi-

nated arm cells, fusoid cells, rosette cells, and collateral

vascular bundles surrounded by a double sheath; and a C3

photosynthetic pathway are features shared by all the

studied species and also are general for Bambusoideae

(Metcalfe 1960; Calderón and Soderstrom 1973; Hattersley

1987; Judziewicz et al. 1999; Oliveira et al. 2008; Gomes

and Neves 2009; Viana 2010; Mota 2013; Leandro et al.

2016b). This study reveals many anatomical similarities

among the sampled species while also demonstrating that

micromorphological features, especially those of the

stomatal complex, prove to be more systematically infor-

mative. Presence or absence of trichomes and their type,

shape of silica bodies, type of arm cells, and the midrib

structure may be useful at the species level.

Fig. 3 Anatomy from light

microscopy of Chusquea

ramosissima informal group

species in cross section. a C.

ramosissima (Hatschbach

42746); b Chusquea sp. nov.

(Uzquiano et al. 10); c C. tenella
(Klein and Bresolin 9477)

showing arm cells with

invaginations from the abaxial

side; d C. tenuiglumis (Clark

and Windisch 864) showing

midrib comprising two

opposing vascular bundles

sharing the same bundle sheath;

e C. tenella (Clark and

Windisch 725) showing midrib

comprising three vascular

bundles (two smaller ones

opposing the major one), all

sharing the same bundle sheath.

Arrowhead = stoma. ac arm

cell; bc bulliform cell, fc fusoid

cell, p phloem, rc rosette cell,

x xylem. Scale bars 5 lm (c),
10 lm (a), 15 lm (b, d, e)

752 T. D. Leandro et al.

123



Implications for systematics

The occurrence of intercostal fibers, presence or absence of

papillae on the subsidiary cells and midrib structure seem

to be the primary differences between the subtribes Chus-

queinae and Arthrostylidiinae (Soderstrom and Ellis 1987;

Leandro et al. 2016b). Intercostal fibers and a simple

midrib are diagnostic features for Arthrostylidiinae,

whereas the Chusqueinae is recognized by the presence of

a stomatal apparatus bearing two papillae per subsidiary

cell, lack of intercostal fibers, and a complex midrib (Fisher

et al. 2009, 2014; Clark et al. 2015). Comparatively,

Guaduinae is distinguished from these two subtribes by the

adaxial epidermis having abundant stomata and often well-

developed papillae (Judziewicz et al. 1999; Ruiz-Sanchez

et al. 2008; BPG 2012; Clark et al. 2015). Guaduinae also

lack intercostal fibers and possess a complex midrib, and

both the Arthrostylidiinae and Guaduinae lack any papillae

on the subsidiary cells (Clark et al. 2015).

Papillae are considered characteristic of bamboo leaves

(Judziewicz et al. 1999), and thus they may be systemati-

cally informative for this group (e.g., Calderón and

Soderstrom 1973; Gomes and Neves 2009; Zhang et al.

2014; Leandro et al. 2016b); however, in our study,

intraspecific variation in the development of papillae is

observed in all four species, in which papillae may vary

from poorly to well developed (rarely completely absent).

For example, C. ramosissima specimens herein studied

exhibit papillae mostly in the stomatal band, as well as on

the adaxial surface, but specimens from Argentina exhibit

an epidermis with no papillae on the long cells of either

surface (Lizarazu 2013; Montti et al. 2008). Despite this

remarkable intraspecific variation, C. ramosissima consis-

tently exhibits a stomatal apparatus bearing two branched

papillae (this study and Lizarazu 2013). Because C.

ramosissima is so widely distributed (Judziewicz et al.

1999), it is perhaps not surprising that it exhibits variation

in some features, but it is also possible that some of this

variation in papillar development in the species of this

group is due to environmental effects (e.g., sun/shade,

March and Clark 2011) that have not been explored in this

group. Also, we observed that papillar development in C.

ramosissima and C. tenella did not correlate with repro-

ductive state—this is consistent with the fact that we also

observed some variation among the specimens of C.

tenuiglumis and Chusquea sp. nov., which only sterile

material was examined.

The stomatal apparatus in Bambusoideae (and Poaceae)

is composed of a pair of guard cells surrounded by two

paracytic subsidiary cells (Metcalfe 1960; Ellis 1979;

Judziewicz et al. 1999). Subsidiary cells bearing two

papillae are a feature shared by all the studied species, and

it is putatively a synapomorphic character for Chusquea

within the woody bamboos, although it is known to occur

in Olyreae (Soderstrom and Ellis 1987; Fisher et al.

2009, 2014; Clark et al. 2015). Although the presence of

branched papillae on the subsidiary cells has been previ-

ously reported for other species of Chusquea (Clark

1986, 1989), the presence of two branched papillae that

completely overarch the guard cells herein observed in C.

ramosissima and C. tenuiglumis is highly informative for

identifying these two species within this group. However,

branched papillae are easily visible only with aid of SEM

techniques, and thus the stomatal apparatus of these species

may be erroneously interpreted. For instance, based on

light microscopy, Montti et al. (2008) reported that the

stomatal apparatus of C. ramosissima bears refractive

papillae, without indicating how many per cell or the

structure of the papillae. Refractive papillae were reported,

but not adequately defined or described, by Soderstrom and

Ellis (1987) for some bamboo groups, but not for Chus-

queinae. It is possible that under LM, the papillae of the

subsidiary cells appear to be refractive, but in the SEM

images (Figs. 1a–i, 2a–c), these papillae do not appear

qualitatively different from the papillae of the long cells.

Species herein studied share a complex midrib vascula-

ture as expected forChusquea (BPG2012; Clark et al. 2015),

although not composed solely of independent vascular

bundles. The complex midrib in species of subg. Rettbergia

(currently 15 described species) often comprises at least two

vascular bundles, each one usually with their own bundle

sheaths (except for C. pulchella L.G. Clark and C. sellowii

Rupr., in which two bundles share both sheaths) (Mota

2013), and Guerreiro et al. (2014) showed this condition in

three species of Chusquea subg. Platonia. In contrast, a

midrib composed of one central vascular bundle that shares

its own bundle sheaths with one or two opposing vascular

bundles is a shared feature among the species herein studied,

which are all placed within subg. Chusquea (Fisher et al.

2009). In theC. ramosissima informal group, a midrib with a

few elements of xylem and phloem embedded in fibers is

observed opposite to the abaxial vascular bundle—and

sometimes these are difficult to distinguish. For instance,

Montti et al. (2008) interpreted the midrib vasculature of C.

tenella as comprising two opposing vascular bundles; how-

ever, two groups of xylem elements can be distinguished in

the figure provided by the authors (see Fig. 6D in Montti

et al. 2008). The developmental pattern of the midrib vas-

culature within Chusquea deserves broader study in order to

verify its value for systematics across the genus.

All species of the C. ramosissima informal group,

including Chusquea sp. nov., share horizontally oriented,

dumbbell-shaped silica cells over the veins, which was also

observed by Montti et al. (2008) and Lizarazu (2013) for C.

ramosissima and C. tenella. Soderstrom and Ellis (1987)

reported that horizontally oriented, dumbbell-shaped silica

Foliar micromorphology and anatomy of the Chusquea ramosissima informal group 753

123



cells over the veins are diagnostic for Chusqueinae and

unusual in bamboos generally, with the more typical con-

dition being vertically oriented silica cells. However,

although horizontally oriented dumbbell-shaped silica cells

over the veins are characteristic of the C. ramosissima

informal group and a few other species (e.g., C. coronalis

Soderstr. & C. Calderón, C. juergensii Hack., C. pittieri

Hack., C. simpliciflora Munro), many other species of

Chusquea show only vertically oriented silica cells over the

veins (Clark 1986, 1990; Lizarazu 2013).

Chusquea ramosissima and C. tenuiglumis exhibit

branched papillae on the subsidiary cells, but they may be

distinguished by the margins (dimorphic or not), and by the

number of rows of stomata in the intercostal zone on the

abaxial surface (two or three). The stomatal apparatus is

also useful for distinguishing C. ramosissima and Chus-

quea sp. nov., which are morphologically similar, although

the relatively wider leaf blades and shorter synflorescences

of Chusquea sp. nov. initially suggested that it might

deserve recognition as a distinct species. Our results sup-

port this, and it is being described as a new species as part

of a taxonomic revision of this group. Chusquea tenella

and Chusquea sp. nov. are similar in their micromorphol-

ogy, but C. tenella differs from it and the other two species

of the group in having three vascular bundles in the midrib

rather than just two in addition to differences in leaf blade

shape and size and spikelet characters.

Conclusions

Our results show that horizontally elongated silica cells

over the veins and adaxial arm cells with invaginations

only from the abaxial side, along with morphological fea-

tures such as culm leaves with pseudopetiolate, deciduous

blades that usually remain green and racemose or weakly

paniculate synflorescences (Fisher et al. 2009, 2014)

strongly support the recognition of the Chusquea ramo-

sissima informal group. The results also support the

recognition of Chusquea sp. nov. from Bolivia as distinct

from C. ramosissima. Broader foliar anatomical and

micromorphological studies within Chusquea are needed,

including study of the midrib structure and the potential for

environmental effects on micromorphology.

Taxonomic treatment

Identification key based on micromorphological

and anatomical features of the Chusquea ramosissima

informal group

1a. Each subsidiary cell bearing two branched papillae

(Fig. 1c, i) ………………………………………… 2

1b. Each subsidiary cell bearing two simple papillae

(Figs. 1f, 2c) ………………………………………… 3

2a. Leaf blade margins dimorphic, often exhibiting groups

of two rows of stomata in the intercostal zone on the

abaxial surface ……………………… C. ramosissima

2b. Leaf blade margins structurally similar; often exhibiting

groups of three rows of stomata in the intercostal zone

on the abaxial surface ………………… C. tenuiglumis

3a. Midrib composed of two opposing vascular bundles

(e.g., Fig. 3e) …………………… Chusquea sp. nov.

3b.Midrib composed of three vascular bundles, two smaller

ones opposing the major one (Fig. 3f) …… C. tenella

General features for the Chusquea ramosissima informal

group

Adaxial surface: Long cells tabular with undulate anti-

clinal walls. Papillae often present on the long cells; often

present on the bulliform cells. Short cells over the veins

and usually scattered between the veins: silica cells saddle

shaped, dumbbell shaped or irregular crenate, horizontally

oriented over the veins, vertically oriented in the intercostal

zone; cork cells usually present, occurring in silico-sub-

erose pairs in the intercostal zone. Prickles often present.

Microhairs often present, unicellular and silicified or

bicellular and non-silicified. Macrohairs rarely present

(observed in Chusquea sp. nov.), unicellular. Stomata

usually absent, if present with subsidiary cells low dome

shaped or low triangular shaped; and two papillae per

subsidiary cell, branched or simple.

Abaxial surface: Long cells tabular with undulate anti-

clinal walls. Papillae often present on the long cells. Short

cells mainly over the veins. Silica cells saddle shaped,

dumbbell shaped, or irregular crenate, horizontally oriented

over the veins, vertically oriented in the intercostal zone;

cork cells usually present, occurring in silico-suberose

pairs in the intercostal zone. Prickles often present. Mi-

crohairs often present, unicellular and silicified or bicel-

lular and non-silicified. Macrohairs usually observed in C.

tenella and Chusquea sp. nov., unicellular. Stomata present

in rows in the intercostal zone, subsidiary cells low dome

shaped or low triangular shaped, two papillae per sub-

sidiary cell, branched or simple.

Cross section: Bulliform cells adaxially present, organized

in fan-shaped arrays. Mesophyll with two layers of asym-

metrically invaginated arm cells adjacent to the adaxial

epidermis, one layer adjacent to the abaxial epidermis;

adaxial arm cells with invaginations from the abaxial side;

abaxial arm cells mostly with invaginations from the

adaxial side. Fusoid cells always present. Vascular bundles

collateral; first and third orders. Midrib complex (com-

prising more than one vascular bundle), all bundles sharing

the same bundle sheaths. Girders always present adjacent

754 T. D. Leandro et al.

123



to the vascular bundles. Margins mainly acute; sometimes

acute and obtuse (dimorphic).

Description of each species including only differences

from the general condition

Chusquea ramosissima: Papillae usually well developed

on both surfaces, but sometimes poorly developed or

absent. Silica cells dumbbell shaped. Prickles absent. Mi-

crohairs on both surfaces; bicellular and non-silicified.

Macrohairs rarely observed on the abaxial surface; uni-

cellular. Stomatal apparatus with subsidiary cells low tri-

angular shaped; two branched papillae per subsidiary cell.

Midrib comprising two opposing vascular bundles. Mar-

gins acute and obtuse (dimorphic).

Chusquea tenella: Papillae well developed on both sur-

faces. Silica cells saddle shaped and dumbbell shaped.

Prickles on both surfaces. Microhairs on the abaxial sur-

face; mostly bicellular and non-silicified, less frequently

unicellular and silicified. Macrohairs sometimes observed

on the abaxial surface; unicellular. Stomatal apparatus

with subsidiary cells low triangular shaped; two simple

papillae per subsidiary cell. Midrib comprising three vas-

cular bundles; two smaller ones opposing the major one.

Margins acute.

Chusquea tenuiglumis: Papillae usually absent from the

long cells, rarely present adaxially. Silica cells saddle

shaped and dumbbell shaped. Prickles absent. Microhairs

rarely observed on the abaxial surface; bicellular, and non-

silicified. Macrohairs absent. Stomatal apparatus with

subsidiary cells low triangular shaped; two branched

papillae per subsidiary cell. Midrib comprising two

opposing vascular bundles. Margins acute.

Chusquea sp. nov.: Papillae well developed on both sur-

faces. Silica cells mostly dumbbell-shaped, and also

irregular crenate. Prickles on both surfaces. Microhairs on

both surfaces; bicellular and non-silicified. Macrohairs on

both surfaces; unicellular. Stomatal apparatus with sub-

sidiary cells low dome shaped; two simple papillae per

subsidiary cell. Midrib comprising two opposing vascular

bundles. Margins acute.

Acknowledgements This research was completed as a partial ful-

fillment for the first author’s scholarship funded by the Coordenação

de Aperfeiçoamento de Pessoal de Nı́vel Superior—CAPES (PDSE

Process Number 99999.003340/2015-05) and the Conselho Nacional

de Desenvolvimento Cientı́fico e Tecnológico—CNPq (GD Process

Number 163550/2012-3). V.L. Scatena was supported by the Con-

selho Nacional de Desenvolvimento Cientı́fico e Tecnológico—CNPq

(Process Number 301692/2010-6). Field work and SEM by L.G.

Clark was supported by U.S. National Science Foundation Grant

BSR-8906340. The authors are grateful to Dr. Harry T. Horner and

Tracey M. Stewart for their assistance and also for access to the

Microscopy and Nano-Imaging Facility at Iowa State University.

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http://dx.doi.org/10.1111/boj.12192

	The contribution of foliar micromorphology and anatomy to the circumscription of species within the Chusquea ramosissima informal group (Poaceae, Bambusoideae, Bambuseae)
	Abstract
	Introduction
	Materials and methods
	Taxon sampling
	Micromorphology (SEM)
	Anatomy (LM)
	Images and terminology

	Results
	Epidermal surfaces (SEM and clearings)
	Cross section

	Discussion
	Micromorphological and anatomical data
	Implications for systematics

	Conclusions
	Taxonomic treatment
	Identification key based on micromorphological and anatomical features of the Chusquea ramosissima informal group
	General features for the Chusquea ramosissima informal group
	Description of each species including only differences from the general condition


	Acknowledgements
	References