Anais da Academia Brasileira de Ciências (2015) 87(4): 2177-2188 (Annals of the Brazilian Academy of Sciences) Printed version ISSN 0001-3765 / Online version ISSN 1678-2690 http://dx.doi.org/10.1590/0001-3765201520150175 www.scielo.br/aabc

Occurrence and evolutionary inferences about Kranz anatomy in Cyperaceae (Poales)

Shirley Martins1, Marccus Alves2 and Vera L. Scatena3

1Centro de Ciências Biológicas e da Saúde, Laboratório de Botânica, Universidade Estadual do Oeste do Paraná, UNIOESTE, Rua Universitária, 2069, Jardim Universitário, 85819-110 Cascavel, PR, Brasil 2Centro de Ciências Biológicas, Departamento de Botânica, Laboratório de Morfotaxonomia Vegetal, Universidade Federal de Pernambuco, UFPE, Rua Prof. Moraes Rego, s/n, Cidade Universitária, 50670-901 Recife, PE, Brasil 3Instituto de Biociências, Departamento de Botânica, Laboratório de Anatomia Vegetal, Universidade Estadual Paulista, UNESP, Avenida 24A, 1515, Bela Vista, 13506-900 Rio Claro, SP, Brasil

Manuscript received on March 5, 2015; accepted for publication on May 13, 2015

ABSTRACT Cyperaceae is an angiosperm family with the greatest diversity of species with Kranz anatomy. Four different types of Kranz anatomy (chlorocyperoid, eleocharoid, fimbristyloid and rhynchosporoid) have been described for this angiosperm family, and the occurrence and structural characteristics of these types are important to trace evolutionary hypotheses. The purpose of this study was to examine the available data on Cyperaceae Kranz anatomy, emphasizing taxonomy, geographic distribution, habitat and anatomy, to infer the potential origin of the Kranz anatomy in this family. The results showed that the four types of

Kranz anatomy (associated with C4 photosynthesis) in Cyperaceae emerged numerous times in unrelated phylogenetic groups. However, the convergence of these anatomical types, except rhynchosporoid, was observed in certain groups. Thus, the diverse origin of these species might result from different environmental pressures that promote photorespiration. Greater variation in occurrence of Kranz anatomy and anatomical

types was observed in Eleocharis, whose emergence of the C4 pathway was recent compared with other genera in the family, and the species of this genus are located in aquatic environments. Key words: anatomy, ultrastructure, taxonomy, phylogeny, Cyperaceae.

INTRODUCTION increased levels of O2 (Sage 2004, Lundgren et al. 2014). Several factors, such as water stress, high The Kranz anatomy represents a structural temperature and lightness, and the salinity and high modification of photosynthetic tissues, generally density of plants, influence the reduction of CO two distinct concentric layers of chlorenchyma 2 (Sage et al. 2012). cells, associated with C metabolism, the main 4 The structural modification (Kranz anatomy) function in plants with suppressed photorespiration, was initially reported in Cyperaceae species induced through decreased levels of CO and 2 (Haberlandt 1914). Since then, several studies using

Correspondence to: Shirley Martins different approaches have examined the Kranz

E-mail: [email protected] structure and the C4 pathway in this family (e.g.

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Carolin et al. 1977, Takeda et al. 1985, Bruhl et al. The variations of Kranz anatomy observed in 1987, Ueno and Samejina 1989, Bruhl 1995, Bruhl this family have generated important taxonomic, and Perry 1995, Soros and Dengler 2001, Martins ecological and evolutive interpretations. Therefore, and Alves 2009, Martins and Scatena 2011). The the aim of the present study was to review Kranz structure varies in the Cyperaceae family, taxonomic, ecological, structural and phylogenetic and four anatomical types have been described: data associated with the structural analyses chlorocyperoid, eleocharoid, fimbristyloid and performed with Cyperaceae species of the four rhynchosporoid. These types anatomically differ Kranz types (Martins and Scatena 2011, Martins in the number and continuity of the vascular and Scatena 2015, Martins et al. 2015) to develop bundle sheath and in the presence of chloroplasts potential hypotheses concerning the origins of in the bundle sheath (Carolin et al. 1977, Soros and these plants. Dengler 2001, Martins and Scatena 2011). MATERIALS AND METHODS Representatives of the Cyperaceae family with Kranz anatomy are restricted to four of The species used to illustrate anatomical and the 13 tribes of the Cyperoideae subfamily: ultrastructural features are listed in Table I. The Abildgaardieae, Cypereae, Eleocharideae and voucher materials were deposited at the Herbarium Rhynchosporeae (Goetghebeur 1998). Among of the Department of Botany, Universidade these, only Abildgaardieae and Eleocharideae Estadual Paulista (HRCB). The data collected here are closely related in phylogenetic studies added to the literature are given in Table II. (Ghamkhar et al. 2007, Muasya et al. 2008). To anatomically illustrate the different Kranz The Kranz Cypereae species present a Kranz types, portions of at least three fully expanded leaf anatomy of the chlorocyperoid type, while the blade or scapes were fixed in FAA 50 and stored Abildgaardieae species present a Kranz anatomy in 70% ethanol (Johansen 1940) and subsequently of the fimbristyloid type (Bruhl and Wilson 2007). dehydrated in an ethyl alcohol series and embedded Chlorocyperoid, eleocharoid and fimbristyloid in Historesin (Leica Historesin Embedding Kit, types have been described in the Eleocharideae tribe Nussloch, Germany) (Feder and O’Brien 1968). (Murphy et al. 2007, Martins and Scatena 2015), Transverse sections of the mid-region were and chlorocyperoid and rhynchosporoid types have obtained using a microtome, and the sections been reported in Rhynchosporeae (Martins and were stained with periodic acid-Schiff’s reagent Scatena 2011, Ueno 2013). and toluidine blue (Feder and O’Brien 1968) and Therefore, it has been suggested that the mounted in Entellan (Merck Darmstadt, Germany). origin of Kranz anatomy emerged four times in The images were obtained using Leica DFC 290 Cyperaceae, one type in each tribe (Soros and digital camera on a Leica DM LB microscope Bruhl 2000). In subsequent studies using molecular using IM50 software. markers, five potential appearances of Kranz To illustrate the ultrastructural features of the anatomy were reported: two types in Eleocharideae four Kranz types, samples from the mid-region of and one type in each of the remaining Kranz at least three fully expanded leaf blades or scapes tribes (Besnard et al. 2009). Phylogenetic studies were fixed in 2.5% glutaraldehyde solution in of Abildgaardieae (Ghamkhar et al. 2007) and 0.1 M phosphate buffer, pH 7.3, for 24 h at 5ºC, Eleocharideae (Roalson et al. 2010) indicated post-fixed with 1% osmium tetroxide in the same multiple origins of C4 photosynthesis. buffer for 1 h at 25ºC, dehydrated with an acetone

An Acad Bras Cienc (2015) 87 (4) EVOLUTIONARY INFERENCES ABOUT KRANZ ANATOMY IN CYPERACEAE 2179 series and embedded in Araldite resin (Machado Abildgaardia hygrophila and Fimbristylis and Rodrigues 2004). Ultrathin transverse sections variegata. This tribe is non-monophyletic, and were obtained using a Diatome diamond knife, representatives of the non-Kranz Arthrostylideae and the sections were subsequently stained with tribe emerge as the sister group of the clade uranyl acetate and lead citrate (Reynolds 1963). Fimbristylis-Abildgaardia-Crosslandia (Fig. The samples were examined using a Philips EM 1e). The Kranz taxa of Abildgaardieae possess 301 transmission electron microscope (MET). the fimbristyloid type, characterized by vascular To illustrate the occurrence of the Kranz bundles surrounded by three sheaths, an outer anatomical types in Cyperaceae, these data sheath (Os), a continuous middle sheath (Ms), were plotted onto cladograms from previously and a discontinuous inner sheath (Is) in the published phylogenetic analyses for each tribe: major bundles, interrupted by only one or two Abildgaardieae (Ghamkhar et al. 2007), Cypereae metaxylem vessels (Fig. 1a-b). The outer bundle (Muasya et al. 2009), Eleocharideae (Roalson et al. sheath originates from the ground meristem, while 2010) and Rhynchosporeae (Thomas et al. 2008). the middle and inner sheaths develop from the procambium. The chloroplasts in the outer-sheath RESULTS cells present well-developed grana, similar to the Abildgaardieae tribe – This tribe has six genera mesophyll cells. The middle-sheath cells do not with Kranz anatomy, Abildgaardia, Bulbostylis, show chloroplasts, and the inner-sheath cells show Crosslandia, Fimbristylis, Nelmesia and Nemum centrifugal chloroplasts without grana (Fig. 1c) and

(Tab. II), and two non-Kranz (C3) species, with convoluted and parallel thylakoids (Fig. 1d).

TABLE I Cyperaceae species used to illustrated the anatomical and ultrastructural features with Kranz types and vouchers, respectively. Tribes and species Kranz anatomical type Voucher Abildgaardieae Bulbostylis conifera L. fimbristyloid S. Martins 329 Bulbostylis scabra (J. Presl. & C. Presl.) C.B. Clarke fimbristyloid S. Martins 408 Fimbristylis autumnalis L. fimbristyloid V.L. Scatena 343 Fimbristylis complanata (Retz.) Link fimbristyloid V.L. Scatena 344 Fimbristylis dichotoma (L.) Vahl fimbristyloid S. Martins 398 Cypereae Cyperus compressus Jacq. chlorocyperoid S. Martins 228 Cyperus ligularis L. chlorocyperoid S. Martins 330 Cyperus maritimus Poir. chlorocyperoid S. Martins 226 Kyllinga brevifolia Rottb. chlorocyperoid S. Martins 288 Pycreus flavescens (L.) Rchb. chlorocyperoid S. Martins 327 Eleocharideae Eleocharis minima Kunth eleocharoid S. Martins 405 Rhynchosporeae Rhynchospora barbata (Vahl) Kunth chlorocyperoid S. Martins 313 Rhynchospora globosa Lindl. rhynchosporoid S. Martins 305 Rhynchospora terminalis Kunth rhynchosporoid S. Martins 302

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TABLE II

Cyperaceae genera with Kranz anatomy (C4 photosynthesis), detach to taxonomical classification, Kranz anatomical types, biochemical subtypes, habitat and distribution (K-C = chlorocyperoid; K-E = eleocharoid; K-F = fimbristyloid; K-R = rhynchosporoid). Tribes and genera (number of C and C Kranz Biochemical 3 4 Habitat Distribution species) type type Abildgaardieae 1,2,3,5,6 Abildgaardia (1 C3/16 C4) K-F NADP-ME Dry field and distubed area Tropical and subtropical 1,2,3,4,5,6 Bulbostylis (150 C4) K-F NADP-ME Dry field, rare moist area Tropical 2,4,6 Crosslandia (4 C4) K-F NADP-ME Dunes and dry field Tropical (Australia) 1,2,3,4,5,6 Fimbristylis (1 C3/300 C4) K-F NADP-ME Dry fields and distubed area Tropical (Asia e Australia) 2,4,6 Nelmesia (1 C4) K-F NADP-ME Field moist Tropical (Africa) 2,4,6 Nemum (10 C4) K-F NADP-ME Field moist Tropical (Africa) Cypereae 2,4,6 Alinula (4 C4.) K-C NADP-ME Field moist Tropical (Africa) 2,4,6 Ascolepis (20 C4) K-C NADP-ME Field moist Tropical 1,2,3,4,5,6 Cyperus subg. Cyperus (260 C4) K-C NADP-ME Field moist and distubed area Tropical, rare subtropical 1,2,3,4,5,6 Kyllinga (75 C4) K-C NADP-ME Field moist and distubed area Tropical, rare subtropical 2,4,6 Lipocarpha (35 C4) K-C NADP-ME Field moist Tropical, rare subtropical 1,2,3,4,5,6 Pycreus (120 C4) K-C NADP-ME Field moist and distubed area Tropical

2,4,5,6 Tropical (Africa, Asia and Queenslandiela (1 C ) K-C NADP-ME Dunes 4 Australia) 2,4,5,6 Remirea (1 C4) K-C NADP-ME Dunes Tropical 2,4,5,6 Sphaerocyperus (1 C4) K-C NADP-ME Dry field Tropical (Africa) 2,3,4,5 Volkiella (1 C4) K-C NADP-ME Dunes Tropical (Africa) Eleocharideae K-E; 1,2,3,4,5,6,8 Eleocharis (244 C3/8 C4) K-C; NAD-ME Border of river and lakes Tropical (America) K-F Rhynchosporeae Rhynchospora grupo Capitatae K-C; 1,2,3,4,5,7 NADP-ME Dry and moist fields Neotropical (5 C3/28 C4) K-R 1Bruhl and Perry (1995), 2Bruhl and Wilson (2007), 3Bruhl et al. (1987), 4Goetghebeur (1998), 5Martins and Scatena (2011), 6Soros and Bruhl (2000), 7Ueno (2013), 8Martins and Scatena (2015).

Cypereae tribe – This tribe possesses 19 Kranz, characterized by two sheaths surrounding genera, 10 of which are Kranz species, including nine the vascular bundles: a continuous outer sheath exclusively Kranz (C4) species (Alinula, Ascolepis, (Os) and a discontinuous inner sheath (Is) in the Kyllinga, Lipocarpha, Pycreus, Queenslandiela, major bundles (Fig. 2a). Both bundle sheaths Remirea, Sphaerocyperus and Volkiella) and in develop from the procambium. The outer-sheath Cyperus restricted to Cyperus subg. Cyperus (Tab. cells do not have chloroplasts (Fig. 2b), and the II). This tribe is monophyletic, and the Kranz taxa inner-sheath cells are generally centrifugal (Fig. emerge in a separate clade from non-Kranz taxa 2b), with inconspicuous grana and convoluted and (Fig. 2e). Cypereae present chlorocyperoid type contorted thylakoids (Fig. 2c-d).

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Figure 1 - Anatomical and ultrastructural features of the Kranz-fimbristyloid type and distribution of this type in the phylogeny of the Abildgaardieae and Arthrotylideae tribes. a-b. Bulbostylis scabra and Fimbristylis complanata, respectively – leaf in transverse section showing the vascular bundle. c-d. Fimbristylis autumnalis. c. Vascular bundle, highlighting centrifugal distribution of chloroplasts. d. Chloroplast of the inner bundle sheath cell with contorted and parelell thylakoids. e. Phylogeny of Abildgaardieae and Arthrotylideae tribes, adapted from Ghamkhar et al (2007). ART = Arthrostylideae; ABI = Abildgaardieae; Ch = chlorenchyma; Is = inner bundle sheath; Ms = middle bundle sheath; Mx = metaxylem; Os = outer bundle sheath; Th = thylakoids. Bars: (a-b) = 20 µm; (c) = 10 µm, (d) = 0.2 µm. For interpretation of the references to color in this figure, the reader is referred to the web version of this article.

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Figure 2 - Anatomical and ultrastructural features of the Kranz-chlorocyperoid type and distribution of this type in the phylogeny of the Cypereae tribe. a. Kyllinga brevifolia – leaf in transverse section showing the vascular bundle. b. Cyperus compressus – vascular bundle, highlighting centrifugal distribution of chloroplasts. c-d. Cyperus maritimus and C. ligularis, respectively – chloroplast of the inner bundle sheath cell with convoluted and contorted thylakoids, respectively. e. Part of phylogeny of Cypereae tribe, detach to Kranz species, adapted from Muasya et al. (2008). Ch = chlorenchyma; Is = inner bundle sheath; Os = outer bundle sheath; Th = thylakoids. Bars: (a) = 20 µm; (b) = 5 µm; (c-d) = 0.5 µm. For interpretation of the references to color in this figure, the reader is referred to the web version of this article.

Eleocharideae tribe – This tribe only includes type shows two continuous vascular bundle sheaths the genus Eleocharis (250 spp.), presenting Kranz (Fig. 3a): an outer sheath (Os) without chloroplasts

(C4) and non-Kranz species (C3) (Tab. II) and and an inner sheath (Is) with chloroplasts that do facultative C3/C4 and intermediate C3-C4 species. not show a constant location and have parallel These species possess leaf lamina reduced to a thylakoids, occasionally forming grana (Fig. 3b- tubular basal sheath, and the scape is the principal c). The fimbristyloid and chlorocyperoid types photosynthetic organ. The Kranz species of in Eleocharis are anatomically similar to those Eleocharis present eleocharoid, fimbristyloid and previously described for Abildgaardieae and chlorocyperoid types (Fig. 3d-e). The eleocharoid Cypereae.

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Figure 3 - Anatomical and ultrastructural features of the Kranz-eleocharoid type and distribution of this type in the phylogeny of the Eleocharideae tribe. a-c. Eleocharis minima. a. Scape in transverse section showing the vascular bundle. b. Vascular bundle, detach to chloroplast without Constant location in the inner bundle sheath. c. Chloroplast with parallel thylakoids in the inner bundle sheath. d-e. Part of phylogeny of the Rhynchosporeae tribe, highlighting the Kranz species, adapted from Roalson et al. (2010). Ch = chlorenchyma; Is = inner bundle sheath; Os = outer bundle sheath; Th = thylakoids. Bars: (a) = 20 µm; (b) = 2 µm; (c) = 0.5 µm. For interpretation of the references to color in this figure, the reader is referred to the web version of this article.

Rhynchosporeae tribe – This tribe possesses (Tab. II). This species presents chlorocyperoid two genera, Pleurostachys and Rhynchospora, and the rhynchosporoid Kranz types that occur in and only Rhynchospora has species with Kranz distinct clades in the phylogenetic analysis (Fig. 4e). anatomy (C4) restricted to Pauciflorae (Pau) and The chlorocyperoid has two sheaths surrounding Pluriflorea (Plu) sections from the Capitatae group the vascular bundles, wherein the outer (Os) is

An Acad Bras Cienc (2015) 87 (4) 2184 Shirley Martins, Marccus Alves and Vera L. Scatena continuous and the inner (Is) is discontinuous In the Cypereae tribe, the Kranz anatomy likely in the major bundles (Fig. 4a). Similar to the derived from a single source, as the Kranz species chlorocyperoid type in Cypereae, both sheaths in this tribe emerged in the same clade (Muasya (outer and inner) originate from the procambium. et al. 2002, 2009) and have the same anatomical The chloroplasts are absent in the outer sheath (chlorocyperoid) and biochemical (NADP- cells (Os), whereas these organelles are centrifugal ME) types and similarities in the structure of the or without pattern, with convoluted and parallel chloroplasts. The emergence of C4 in this tribe is thylakoids in the inner sheath cells (Fig. 4b). The indicated ca. 10.9 ± 3.8 Ma (Besnard et al. 2009), rhynchosporoid present a unique continuous bundle initially in mesophyte species (with C3 mesophytes sheath (Ss – single sheath) (Fig. 4c), originating ancestors) and subsequently in xerophytes (with from the procambium and presenting cells with C4 mesophytes ancestors), as most of the C4 chloroplasts located centrifugally or without species primarily occur in mesic environments and pattern, with inconspicuous grana and parallel a minority of these species are observed in xeric thylakoids (Fig. 4d). areas. This hypothesis has also been postulated for

the C4 species of Poaceae (Osborne and Freckleton DISCUSSION 2009). The occurrence of C4 metabolism in mesic environments with high water availability is The data obtained from the present study and associated with the high density, high temperature previously published studies suggest that multiple and increased luminosity, which reduce the origins of Kranz anatomy occurred in Cyperaceae absorption of CO2 and carbohydrate production in due to different environmental factors that promote plants (Bowes et al 200, Sage et al. 2012). photorespiration. The Kranz anatomy is uniform in The plasticity of the C4 pathway makes it Abildgaardieae Cypereae tribes, showing only one difficult to interpret the origin of this pathway in anatomical type, and varied in Rhynchosporeae and Eleocharideae, with the possibility of two sources Eleocharideae tribes, showing more than one type. with reversals for C3 or multiple origins. The Kranz In Abildgaardieae, the Kranz anatomy (C4) types in this tribe vary between individuals of the likely had only one origin, before the occurrence of same species (Ueno 1996, 2004, Murphy et al. a single anatomical (fimbristyloid) and biochemical 2007) and even in the same individual (Martins and

(NADP-ME) type, with possible reversion to the C3 Scatena 2015). As eleocharoid types are considered pathway (not Kranz) in Abildgaardia hygrophila, more related to C4 metabolism, and the fimbristyloid Fimbristylis variegata and representatives of the types are considered C3-C4 intermediates (Murphy Arthrostylideae tribe. This hypothesis is confirmed et al. 2007), it is likely that the aquatic environment by phylogenomic and phylogenetic studies (Besnard of these plants promotes anatomical plasticity, et al. 2009, Ghamkhar et al. 2007), suggesting the such as the number of vascular bundles and the initial onset of C4 photosynthesis in Bulbostylis (ca. presence and development of aerenchyma (Martins 19.6 ± 4.9 Ma), followed by Fimbristylis (ca. 12.3 and Scatena 2015), as observed in other anatomical ± 3.8 Ma). Moreover, the species of Abildgaardieae structures. is primarily observed in xeric environments (open Structurally, the difference between the fields, rocky outcrops, dunes), and the 4C pathway eleocharoid and fimbristyloid types is the size and must have arisen in this environment, where this shape of the cells of the inner layer chlorenchyma biochemical pathway is more advantageous (Sage (Ch) (both originate from the ground meristem), 2004, Sage et al. 2012). which is slightly larger and elongated in the first

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Figure 4 - Anatomical and ultrastructural features of the Kranz-chlorocyperoid and Kranz-rhynchosporoid types and distribution of these types in the phylogeny of the Rhynchosporeae tribe. a-b. Rhynchospora barbata. a. Leaf in transverse section showing the vascular bundle. b. Chloroplast of the inner bundle sheath cell with parallel thylakoids. c-d. Rhynchospora globosa. c. Leaf in transverse section showing the vascular bundle. d. Chloroplast of the inner bundle sheath cell with parallel thylakoids. e. Part of phylogeny of Rhynchosporeae tribe, detach to Kranz species, adapted from Thomas et al. (2008). Ch = chlorenchyma; Is = inner bundle sheath; Os = outer bundle sheath; Pau = Pauciflorae; Plu = Pluriflorae; Ss = single bundle sheath; Th = thylakoids. Bars: (a, c) = 20 µm; (b, d) = 0.5 µm. For interpretation of the references to color in this figure, the reader is referred to the web version of this article.

An Acad Bras Cienc (2015) 87 (4) 2186 Shirley Martins, Marccus Alves and Vera L. Scatena type, and rounded and smaller in the second type. (Ueno and Koyama 1987), living sympatrically

These types also differ in the continuity of the with non-Kranz (C3) species of the same genus. inner sheath in the major vascular bundles, being Thus, it has been suggested that the C4 pathway continuous only in the eleocharoid type (Soros arose in some lineages of the genus as an adaptation and Bruhl 2000). However, the continuity of the to conditions of water stress, high temperatures and inner sheath might vary along the scape and can light from C3 ancestors potentially occurring in this be interrupted in the apical region, as indicated for type of environment. However, the occurrence of Eleocharis minima (Martins and Scatena 2015), different Kranz types in closer phylogenetic groups described as Kranz eleocharoid (Murphy et al. living sympatrically is intriguing and requires more 2007, Martins and Scatena 2011). detailed studies.

In Eleocharideae, the C4 pathway is considered The variations among the Kranz types in intermediate to recent (Eleocharis vivipara - ca. Cyperaceae reflect essential differences in the 10.5 ± 3.2 Ma; E. baldwinii - 4.4 ± 2.1 Ma) compared structure, not the origin, of the tissues. The with the other C4 species of this family (Besnard et homologous chlorophyllian cells surrounding the al. 2009). These authors indicated that Cyperaceae vascular bundles arise from the ground meristem species with different Kranz types do not present and might have an elongated (chlorophyll significant genetic modifications. Thus, before the parenchyma) or rounded (outer sheath) shape. plasticity observed in Eleocharis, the changing of In chlorocyperoid, eleocharoid and fimbristyloid

C3 to C4 metabolism might have required a small types, the procambium gives rise to two vascular number of genetic modifications, as indicated by sheaths, with chloroplasts only in the inner sheath. Christin et al. (2007) for groups with high plasticity In the rhynchosporoid type, only one sheath has in present C4 photosynthesis. Plasticity in Kranz procambial origin and presents chloroplasts. This anatomy was also observed in other C4 groups, sheath is homologous to the outer sheath, which such as some species of Poaceae and Asteraceae lacks chloroplasts with procambial origin. (Araus et al. 1990, Gong et al. 2011). Herein, we showed the multiple origins In Rhynchosporeae, it is likely that the Kranz of Kranz anatomy in Cyperaceae in different anatomy arose twice, once for each anatomical phylogenetic groups, confirming the results of type, as chlorocyperoid and rhynchosporoid occur Soros and Bruhl (2000), Muasya et al. (2002, in distinct clades (Thomas et al. 2008). Both Kranz 2008, 2009), Ghamkhar et al. (2007), Thomas et al. types occur in Pauciflorae and Pluriflorae sections (2008) and Roalson et al. (2010). Thus, this study (Ueno and Koyama 1987), and although these types represents adaptive convergence, as phylogenetic differ in the number of sheaths and chloroplast relationships are not reflected. In addition, the locations, similarities are observed in chloroplast anatomical types are not associated with specific distribution and thylakoid organization. Genetic environmental conditions because the Kranz markers indicate that the C4 pathway is recent in species of Abildgaardieae, Rhynchospora and some Rhynchospora (ca. 7.4 ± 2.8 Ma) compared with the of Cypereae occur in similar xeric environments or other species of this family (Besnard et al. 2009), even live sympatrically and present different Kranz and phylogenetic data show the occurrence of the types. In addition, Eleocharis representatives vary

C4 pathway in derivative gender groups (Thomas in relation to the occurrence and the type of Kranz et al. 2008). The Kranz (C4) of the Rhynchospora anatomy in individuals of the same species. species are primarily Neotropical, occurring in xeric Thus, the Kranz types in Cyperaceae likely environments, such as open fields and savannas result from ecological pressures that promote

An Acad Bras Cienc (2015) 87 (4) EVOLUTIONARY INFERENCES ABOUT KRANZ ANATOMY IN CYPERACEAE 2187 photorespiration, even in different environmental família e as espécies deste gênero vivem em ambientes conditions. The Kranz species of Abildgaardieae aquáticos. and Rhynchosporeae tribes are xerophytes, whereas Palavras-chave: anatomia, ultraestrutura, taxonomia, the Cypereae species are mesophytes or xerophytes filogenia, Cyperaceae. and the Eleocharideae species are hydrophytes. Thus, independent of the environment, distinct REFERENCES lineages of Cyperaceae have similar evolutionary Araus JL, Brown RH, Bouton JH and Serret MD. 1990. ), pathways for more efficient photosynthesis. Leaf anatomical characteristics in Flaveria trinervia (C4 Flaveria brownii (C4-like) and their F1 hybrid. Photosynth Res 26: 49-57. ACKNOWLEDGMENTS Besnard G, Muasya AM, Russier F, Roalson NS and

Christin PA. 2009. Phylogenomics of C4 photosynthesis We thank the Fundação de Amparo à Pesquisa do in sedges (Cyperaceae): Multiple appearances and genetic Estado de São Paulo (FAPESP) for a PhD grant convergence. Mol Biol Evol 26: 1909-1919. (2008/09380-2) to S. Martins and financial support Bowes G, Rao SK, Estavillo GM and Reiskind JB. 2002. C mechanisms in aquatic angiosperms: comparisons with (2011/18275-0) and the Conselho Nacional de 4 terrestrial C4 system. Func Plant Biol 29: 379-392. Desenvolvimento Científico e Tecnológico (CNPq) Bruhl JJ. 1995. Sedge genera of the world: relationships and for financial support to V.L. Scatena (301692/2010- new classification of the Cyperaceae. Aust Syst Bot 8: 6 and 471837/2011-3) and to S.R. Machado 125-305. Bruhl JJ and Perry S. 1995. Photosynthetic pathway- (2008/301464-1) and the Centre of Microscopy of related ultrastructure of C3, C4 and C3-C4 intermediate the Universidade Estadual Paulista (Botucatu). sedge (Cyperaceae), with special reference to Eleocharis. Aust J Plant Physio 22: 521-530. Bruhl JJ, Stone NE and Hattersley PW. 1987. C RESUMO 4 acid decarboxylation enzymes and anatomy in sedges Cyperaceae é uma das famílias de angiospermas com (Cyperaceae): first record of NAD-malic enzyme species. Aust J Plant Physio 22: 521-530. maior diversidade de espécies com anatomia Kranz. Bruhl JJ and Wilson KL. 2007. Towards a comprehensive Para a família foram descritos quatro diferentes tipos survey of C3 and C4 photosynthetic pathway in de anatomia Kranz (clorociperoide, eleocaroide, Cyperaceae. In: Columbus JT, Friar EA, Hamilton CW, fimbristiloide e rincosporoide) e a ocorrência eas Porter JM, Prince LM and Simpson MG (Eds), Monocots: características estruturais desses tipos são importantes comparative biology and evolution, Claremont: Rancho para traçar hipóteses evolutivas. Este trabalho visou Santa Ana Botanic Garden, p. 99-148. Carolin RC, Jacobs Swl and Vesk M. 1977. The examinar dados disponíveis sobre anatomia Kranz de ultrastructure of Kranz cells in the family Cyperaceae. Bot Cyperaceae com ênfase na taxonomia, distribuição Gaz 138: 413-419. geográfica, habitat e anatomia para inferir a possível Christin PA, Salamin N, Savolainen V, Duvall MR origem da anatomia Kranz na família. Verificou-se and Besnard G. 2007. C4 photosynthesis evolved in que os quatro tipos de anatomia Kranz (relacionada à grasses via parallel adaptive genetic changes. Curr Biol 17: 1241-1247. fotossíntese C ) em Cyperaceae surgiram diversas vezes 4 Feder N and O’Brien TP. 1968. Plant microtechnique: some em grupos não relacionados filogeneticamente. Por principles and new methods. Am J Bot 55: 123-142. outro lado, observou-se a convergência desses tipos Ghamkhar K, Marchant AD, Wilson KL and Bruhl JJ. anatômicos, exceto o rincosporoide, em determinados 2007. Phylogeny of Abildgaardieae (Cyperaceae) inferred grupos. Assim, a origem diversa dessas espécies from ITS and trnL-F data. Aliso 23: 149-164. pode ser resultado de diferentes pressões ambientais Goetghebeur P. 1998. Cyperaceae. In: Kubitzki K, Huber H, Rudall PJ, Stevens PS and Stützel T (Eds), The families que promovem a fotorrespiração. Maior variação da and genera of vascular plants. Berlin: Springer-Verlag, p. ocorrência da anatomia Kranz e de tipos anatômicos 141-190. foi observada em Eleocharis, cujo surgimento da via Gong CM, Bai J, Deng JM, Wang GX and Liu XP. 2011.

C4 é recente quando comparado aos demais gêneros da Leaf anatomy and photosynthetic carbon metabolic

An Acad Bras Cienc (2015) 87 (4) 2188 Shirley Martins, Marccus Alves and Vera L. Scatena

characteristics in Phragmites communis in different soil Reynolds ES. 1963. The use of lead citrate at high pH as an water availability. Plant Ecol 211: 675-687. electron-opaque stain in electron microscopy. J Cell Biol Haberlandt Gfj. 1914. Physiological plant anatomy. 17: 208-212. London, MacMillan. Roalson EH, Hinchliff CE, Trevisan R and Silva Johansen D. 1940. Plant microtechnique. New York, Crm. 2010. Phylogenetic relationships in Eleocharis

McGraw-Hill Book Co. Inc. (Cyperaceae): C4 Photosynthesis origins and patterns of Lundgren MR, Osborne CP and Christin PA. 2014. diversification in the spikerushes. Syst Bot 35: 257-271.

Deconstructing Kranz anatomy to understand C4 evolution. Sage RF. 2004. The evolution of C4 photosynthesis. New J Exp Bot 65: 3357-3369. Phytologist 161: 341-370. Machado SR and Rodrigues TM. 2004. Anatomia e ultra- Sage RF, Sage TL and Kocacinar F. 2012. Photorespiration

estrutura do pulvino primário de Pterodon pubescens and the evolution of C4 photosynthesis. Ann Rev Plant Biol Benth. (Fabaceae-Faboideae). Rev Bras Bot 27: 135-147. 63: 19-47. Martins S and Alves M. 2009. Anatomical features of Soros CL and Bruhl JJ. 2000. Multiple evolutionary

species of Cyperaceae from northeastern Brazil. Brittonia origins of C4 photosynthesis in the Cyperaceae. In: Wilson 61: 189-200. KL and Morrison DA (Eds), Monocots: systematics and Martins S and Scatena VL. 2011. Bundle sheath ontogeny evolution. Melbourne: CSIRO Publishing, p. 629-636. in Kranz and non-Kranz species of Cyperaceae (Poales). Soros CL and Dengler NG. 2001. Ontogenetic derivation

Aust J Bot 59: 554-562. and cell differentiation in photosynthetic tissues of C3 and

Martins S, Machado SR and Scatena VL. 2015. An C4 Cyperaceae. Am J Bot 88: 992-1005. overview about ultrastructure of Kranz anatomy in Takeda T, Ueno O, Samejima M and Ohtani T. 1985. An

Cyperaceae (Poales). Rodriguésia 66: 633-643. investigation for the occurrence of C4 photosynthesis in the Martins S and Scatena VL. 2015. Anatomical variations in Cyperaceae from Australia. Bot Mag Tokyo 98: 393-411. scapes of Eleocharis minima Kunth (Cyperaceae, Poales) Thomas T, Araújo AC and Alves MV. 2008. A preliminary – amphibian and Kranz species. Rodriguésia 66: 627-631. molecular phylogeny of the Rhynchosporeae (Cyperaceae). Muasya AM, Simpson DA and Chase MW. 2002. Bot Rev 75: 22-29. Phylogenetic relationships in Cyperus L. s.l. (Cyperaceae) Ueno O. 1996. Structural characterization of photosynthetic inferred from plastid DNA sequence data. Bot J Linn Soc cells in an amphibious sedge, Eleocharis vivipara, in

138: 145-153. relation to C3 and C4 metabolism. Planta 199: 382-393.

Muasya AM, Simpson DA, Verboom GA, Goetghebeur Ueno O. 2004. Environmental regulation of C3 and C4 P, Naczi Rfc, Chase MW and Smets E. 2008. differentiation in the amphibious sedge, Eleocharis Phylogeny of Cyperaceae based on DNA sequence data: baldwinii and comparisons with related species. Plant Cell Current progress and future prospects. Bot Rev 75: 2-21. Environ 27: 627-639. Muasya AM, Vrijdaghs A, Simpson DA, Chase MW, Ueno O. 2013. Ultrastructure and carbon isotope ratios of

Goetghebeur P and Smets E. 2009. What is a genus in leaves in C4 species of Rhynchospora (Cyperaceae) that Cypereae: phylogeny, character homology assessment and differ in the location of Kranz cells. Int J Plant Sci 174: generic circumscription in Cypereae. Bot Rev 75: 52-66. 702-709. Murphy LR, Barroca J, Franceschi VR, Lee R, Ueno O and Koyama T. 1987. Distribution and evolution

Roalson EH, Edwards GE and Ku Msb. 2007. of C4 syndrome in Rhynchospora (Rhynchosporeae-

Diversity and plasticity of C4 photosynthesis in Eleocharis Cyperaceae). Bot Mag Tokyo 100: 63-85. (Cyperaceae). Func Plant Biol 34: 571-580. Ueno O and Samejina M. 1989. Structural features of NAD-

Osborne CP and Freckleton RP. 2009. Ecological malic enzyme type C4 Eleocharis: An additional report of

selection pressures for C4 photosynthesis in the grasses. C4 acid decarboxylation types of the Cyperaceae. Bot Mag Proc Roy Soc Lond B Biol Sci 276: 1753-1760. Tokyo 102: 393-402.

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