Taxonomic Revision of the Dioscorea campestris Species Assemblage (Dioscoreaceae) Author(s): Ricardo S. Couto, Vitor Tenorio, Fernanda da C. Alzer, Rosana C. Lopes, Ricardo C. Vieira, Cláudia B. F. Mendonça, Vania Gonçalves-Esteves, and João Marcelo Alvarenga Braga Source: Systematic Botany, 39(4):1056-1069. 2014. Published By: The American Society of Plant Taxonomists URL: http://www.bioone.org/doi/full/10.1600/036364414X683895 BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder. BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. Systematic Botany (2014), 39(4): pp. 1056–1069 © Copyright 2014 by the American Society of Plant Taxonomists DOI 10.1600/036364414X683895 Date of publication September 3, 2014 Taxonomic Revision of the Dioscorea campestris Species Assemblage (Dioscoreaceae) Ricardo S. Couto,1,4 Vitor Tenorio,2 Fernanda da C. Alzer,2 Rosana C. Lopes,2 Ricardo C. Vieira,2 Cláudia B. F. Mendonça,1 Vania Gonçalves-Esteves,1 and João Marcelo Alvarenga Braga3 1 Museu Nacional, Universidade Federal do Rio de Janeiro, Quinta da Boa Vista s. n., São Cristovão, 20940–040 Rio de Janeiro, Rio de Janeiro, Brazil. 2 Departamento de Botânica, Universidade Federal do Rio de Janeiro, Rua Prof. Rodolpho Paulo Rocco s/n, CCS, 21941–490 Rio de Janeiro, Rio de Janeiro, Brazil. 3 Instituto de Pesquisas Jardim Botânico do Rio de Janeiro, Rua Pacheco Leão, 915, 22460–030 Rio de Janeiro, Rio de Janeiro, Brazil. 4 Author for correspondence (rsscouto@gmail.com) Abstract—The Dioscoreaceae are widely distributed in the tropics, but despite the diversity and utility of the taxa of this family as food (e.g. yam) they are often poorly delimited morphologically and have complicated taxonomic issues. The Dioscorea campestris assemblage comprises 11 names in the literature, with considerable morphological variation among herbarium specimens. Macro- and micromorphological examinations of the tubers, stems, and pollen indicated the recognition only of D. campestris and the proposal of a new combination— Dioscorea pedalis; the other nine names are reduced to synonyms of D. campestris. We report here the pollinic, anatomical, and morphological characters of D. campestris and D. pedalis, and describe the ecology, phenology, geographical distributions, etymology, conservation status, and taxonomies of both species. Keywords—Brazil, Dioscorea pedalis, Dioscoreales, Neotropics, new combination, tuber. The pantropical family Dioscoreaceae comprises approximately 650 species (Govaerts et al. 2007). Usually vines, they are found in many habitats, although mainly in tropical forests. Beyond classical works, such as those of Vellozo (1831, 1881), Grisebach (1842), Hauman (1916), and Knuth (1924), studies of Brazilian Dioscoreaceae have largely been limited to species lists (e.g. Kirizawa 1997) and regional floras (e.g. Kirizawa and Romanini 2003). Taxonomic revisions and studies of species boundaries have been rare. As such, several taxonomic assemblages in the family can be identified, but without specific delimitations (Pedralli 1998; Couto 2010), thus indicating the necessity for more research focusing on the family. Since the first description of Dioscorea campestris Grisebach (1842), several taxa with similar characteristics, as well as varieties and forms of this species, have been described (e.g. Kunth 1850; Hauman 1916; Knuth 1917, 1924). Many of these taxa were later treated as synonyms of D. campestris (e.g. Barroso et al. 1974; Pedralli 1998, 2004; Govaerts et al. 2007; Kirizawa et al. 2010, 2013; The Plant List 2013). The history of this assemblage, combined with the lack of subsequent taxonomic studies, has resulted in a group of herbarium specimens being identified as D. campestris— although they show great morphological diversity. Pedralli (1998, 2004), Govaerts et al. (2007), and The Plant List (2013) treated a total of 16 names as synonyms of D. campestris: D. adenantha Uline, D. campestris Kunth ex Pedralli, D. campestris var. grandiflora Griseb., D. campestris f. longispicata Hauman, D. campestris f. paraguayensis R. Knuth, D. campestris var. parviflora Griseb., D. campestris f. pedalis Uline ex R. Knuth, D. campestris f. piedadensis Uline ex R. Knuth, D. campestris f. plantaginifolia Uline ex R. Knuth, D. campestris f. stenorachis Uline ex R. Knuth, D. loefgrenii R. Knuth, D. nutans R. Knuth, D. sanpaulensis R. Knuth, D. tubuliflora Uline ex R. Knuth, D. tubulosa Griseb., and Helmia campestris Kunth. These taxa have been considered part of the D. campestris assemblage, although Kirizawa et al. (2010, 2013) treated six of these species as valid and distinct from D. campestris (including D. adenantha, D. loefgrenii, D. nutans, D. sanpaulensis, D. tubuliflora, and D. tubulosa). Kirizawa et al. (2010, 2013) considered these taxa to be distinct species as they show characteristics different from those of D. campestris, primarily based on floral and fruit features. These authors also reported that these species are endemic to southeastern and southern Brazil (mainly the states of Minas Gerais and São Paulo), which are considered outside the normal range of the D. campestris assemblage. Our lack of information about the species of Dioscoreaceae, especially those included in taxonomic assemblages of the large genus Dioscorea, complicates species delimitations within the family. The present work therefore proposes an updated taxonomy for the D. campestris assemblage using macro- and micro-morphological data, focusing on traits related to the tubers, stem anatomy, and palynology – which have rarely been used in taxonomic treatments in Dioscorea – thereby considerably amplifying the tools used for delimiting Dioscorea species. Materials and Methods Morphology—Dried specimens were examined at the B, BR, C, CAY, CESJ, COAH, COL, CR, CTES, CUVC, CVRD, ESA, F, FCAB, FURB, GUA, HAL, HAS, HB, HCF, HMUC, HRCB, HST, HUCP, HUEFS, HUPG, HVASF, HXBH, IAC, ICN, INPA, IPA, IRBR, JE, JVR, K, L, LPS, M, MBM, MEXU, MG, MNHN, MO, MVFA, MVFQ, MVM, NY, OPUR, P, PACA, PEL, PH, RB, RBR, RFA, RFFP, S, SMDB, SP, SSUC, U, UFP, UFPR, ULS, UNR, UPCB, US, UV, WU, XAL, Z, and ZT herbaria. The collections by R. S. Couto & L. J. T. Cardoso 265, 266, 267, and R. S. Couto et al. 232, 233, 315, 375, 380, 471, 531, 587, 592, 593 were maintained naturally or were liquidpreserved in FPA (Formalin, Propionic Acid, 50% Alcohol; Ruzin 1999) for later analyses. In order to improve delimitations of the species treated here, we adopted the concepts of de Queiroz (2007) using population aggregation analysis based on combinations of morphological character states, as presented by Davis and Nixon (1992). Anatomy—Three specimens each of D. campestris and D. pedalis were analyzed in terms of their stem anatomy. One specimen of each species was chosen for illustration, and is indicated by an asterisk (*) after the collector’s name in Table 1. The samples were fixed and stored in 70% ethanol (Johansen 1940). Anatomical studies were performed using light microscopy. Stem samples were processed by routine PEG embedding (Burger and Richter 1991). Transverse sections 15–20 mm thick 1056 2014] COUTO ET AL.: REVISION OF DIOSCOREA CAMPESTRIS 1057 Table 3. Measurements (mm) of pollen grains of Dioscorea species (Dioscoreaceae) in equatorial view (n = 10; x = arithmetic mean) Table 1. Vouchers of the materials used in the pollen and anatomical analyses of each species. Samples marked with (*) represent reference material chosen for statistical treatment and illustrations. Polar diameter (PD) D. campestris Pollen Anatomy D. pedalis M. C. Pinheiro 153 (R) R. C. Forzza 4384 (RB) R. S. Couto et al. 232 * (RFA) R. C. Forzza 4384 (RB) R. S. Couto et al. 232 (RFA) R. S. Couto 375 * (RFA) R. S. Couto et al. 593 (RB) R. S. Couto and L. J. T. Cardoso 266 (RFA) R. S. Couto et al. 587 * (RFA) R. S. Couto and L. J. T. Cardoso 266 (RFA) R. S. Couto et al. 593 (RB) R. S. Couto and L. J. T. Cardoso 267 * (RFA) Equatorial diameter (ED) Species Range of variation x Range of variation x D. campestris D. pedalis 12.5–15 15–25 14.2 20.3 17.5–20 25–30 19.6 27 parenchymatous pith, whereas D. campestris has reduced and sclerified pith. Dioscorea pedalis has two peripheral vascular bundles with only one large vessel, while the other taxa have paired vessels. In D. campestris, all of the vascular bundles have pairs of large vessels. Both species form storied cork; however, only D. campestris has brachysclereid layers in this tissue (Fig. 2). In spite of the taxonomic problems common in Dioscorea, analysis of the 11 names forming the D. campestris assemblage demonstrated that D. campestris and D. pedalis were two distinct species (Table 4) and that the remaining taxa should be subsumed under the former species (see Notes below for justification). Dioscorea campestris and D. pedalis can be differentiated by features related to their external morphologies, anatomy, and palynology (Table 4). In particular, D. pedalis can be morphologically distinguished by its very small branches (15–35 cm long), subcoriaceous leaves with petioles strongly canaliculate, short staminate inflorescences (3.3–6.5 cm long), campanulate perianth of staminate flowers with oval and patent lobes, pistillate flowers with filiform branches at the end of each style, and elliptical, papyraceous capsules with smooth margins. D. campestris, on the other hand, is a perennial plant with mediumsized branches (30–250 cm long), chartaceous leaves with flattened petioles slightly canaliculate, relatively long staminate inflorescences (6.5–25 cm long), campanulate perianth of staminate flowers with lobes oblong to obovate and slightly reflexed, pistillate flowers with rounded branches at the end of each style, and oblong, chartaceous capsules with dilated margins. Dioscorea pedalis has an underground system consisting of a rhizomatous tuber with many meristematic points from which aerial stems emerge, whereas D. campestris has an underground system consisting of a discoid to lobed tuber with only one meristematic point (from which the stem emerges). were obtained using a rotary microtome, stained with safranin and astra blue (Bukatsch 1972), and mounted in Canada balsam. Palynology—The 60% lactic acetolysis method was used to prepare specimens for light microscopic analysis (Raynal and Raynal 1971). Photomicrographs of pollen grains were obtained using a Sony Cyber– Shot DSC–W7 digital camera coupled to a Zeiss Axiostar Plus microscope. The pollen grains were measured and photomicrographed within three days of preparation to avoid deformation (Wanderley and Melhem 1991). Non–acetolyzed pollen grains were spread onto the surface of doublesided carbon tape on an aluminum stub, transferred to a vacuum chamber, coated with a thin layer of palladium gold (ca. 2 mm thick), and subsequently observed using a Zeiss DS M960 scanning electron microscope (SEM) at the Cellular Ultrastructure Laboratory of the Biophysics Institute— UFRJ. Three specimens of each species were analyzed and compared; one specimen of each species was chosen for statistical treatment and illustration, and is indicated by an asterisk (*) after the collector’s name in Table 1. Ten measurements of the same type were made of pollen grains from another collection, to confirm the stability of the reference material data (treated hereafter as the comparison material) (SalgadoLabouriau 1973). The pollen grains were distributed onto at least three slides to homogenize the samples (Labouriau et al. 1963). Polar diameter (PD) and equatorial diameter (ED) were measured in equatorial view. The largest equatorial diameter (LED) and smallest equatorial diameter (SED) were measured in polar view, and ten measurements were made of the colpi and exine layers. The terminology used to describe all shapes, sizes, and ornamentation was based on Punt et al. (2007). Results Palynological analyses indicated that the species studied here can be separated based on pollen grain size (small in D. campestris, medium-sized in D. pedalis; Tables 2, 3) and on details of their sexine ornamentation [D. pedalis: lumens frequently with interior beads and small lumens surrounding larger more conspicuous lumens; D. campestris: lumens infrequently with interior beads, with more homogeneous lumen sizes (Fig. 1). Anatomical data indicated significant differences in pith composition, vasculature, and storied cork features between D. pedalis and D. campestris in both their aerial stems and tubers (Table 4). For example, Dioscorea pedalis has wide, Taxonomic Treatment DIOSCOREA CAMPESTRIS Griseb. in Martius et al. Fl. bras. 3(1): 30. 1842. Helmia campestris (Griseb.) Kunth, Enum. Pl. 5: 425. 1850.—TYPE: BRAZIL. “Habitat in provinviae Minarum Generalium campis editis, ubi inter herbas Table 2. Measurements (mm) of pollen grains of Dioscorea species (Dioscoreaceae) in polar view (n = 25; x ± s = arithmetic mean ± standard deviation, CI = 95% confidence interval) Largest equatorial diameter (LED) Smallest equatorial diameter (SED) Species Range of variation x±s CI Range of variation x±s CI D. campestris D. pedalis 17.5–25 20–30 20.5 ± 0.5 27.2 ± 0.5 19.5–21.5 26.2–28.2 10–15 15.0–22.5 13.2 ± 0.3 17.9 ± 0.5 12.6–13.8 16.9–18.9 1058 SYSTEMATIC BOTANY [Volume 39 Fig. 1. Photomicrographs and electron micrographs of Dioscorea. A–B. Dioscorea campestris. A. Distal polar view (SEM). B. Detailed view of the surface (SEM). Arrow indicates granules. C–F. Dioscorea pedalis. C. Optical section representing the distal polar view. D. Distal polar view (SEM). E. Equatorial view, general appearance. F. Detailed view of the surface (SEM). Arrow indicates smaller lumina around the larger lumina. Scale bars: A, C, D, E = 5 mm; B, F = 2 mm. scandit”, s. d., C. F. P. Martius 40, (lectotype: M!; isolectotype: K, designated by Pedralli, 2002). s. d., F. Sellow 50 (lectotype: M!; isolectotype: P! and B!, here designated). Dioscorea campestris var. grandiflora Griseb. in Martius et al. Fl. bras. 3(1): 30. 1842.—TYPE: BRAZIL. Minas Gerais, Dioscorea campestris var. parviflora Griseb. in Martius et al. Fl. bras. 3(1): 30. 1842.—TYPE: BRAZIL. Minas Gerais: 2014] 1059 A comparison of the primary morphological characters differentiating D. campestris and D. pedalis. D. campestris Character Distribution + Pollen grain Pollen grain aperture D. pedalis Argentina, Paraguay, and Brazil, several habitats Small size (20.5 mm) Monocolpate with long and wide colpi (ca. 17.2 4 mm) Columellae with low muri and sparsely perforate, with granules within the lumen, lumina of more uniform size Surface ornamentation Tepal length (mm) Branch length (cm) Inflorescence length (cm) Leaf texture Tepal form and orientation Tuber 1.5–2 30–250 6.5–25 Chartaceous Oblong to obovate, slightly reflexed Discoid to lobed, with only one meristematic point from where the aerial stem emerges Life cycle Capsule Medulla of the aerial stem Vascular bundles of the aerial stem Perennial Oblong, chartaceous, with dilated edge Extremely low and lignified All bundles with one pair of large diameter vessels Storied cork Brachysclereids present Dioscorea campestris f. stenorachis Uline ex R. Knuth, Notizbl. Königl. Bot. Gart. Berlin 7(65): 187. 1917.—TYPE: BRAZIL. Rio de Janeiro: Rio de Janeiro, 1883, A. F. M. Glaziou 14351 (lectotype: P00748327!, here designated; isotypes: C!, G!, P!). Dioscorea campestris f. paraguayensis R. Knuth, Feddes Repert. 22: 346. 1926.—TYPE: PARAGUAY. ”no information of the exact location”, s. d., J. D. Anisits s. n. (holotype: B!; isotype: S). Dioscorea campestris Kunth ex Pedralli. 2004. Diosocoreáceas. Pp. 36 in Flora ilustrada Catarinense, fasc. Diosc., ed. R. Reitz, “pro syn.” Left-twining vine, perennial, dioecious (Figs. 3E; 4A). Tuber discoid to lobed, with only one meristematic point to which the aerial stem is attached, with many fine roots emerging from edge of the tuber, periderm brown–grayish, parenchyma yellowish–white, ca. 15 cm below the soil surface (Figs. 3L–M; 4B–C). Stems to at least 2.5 m long, initially erect to twining, glabrous, terete, unarmed, green, + + + Dioscorea campestris f. plantaginifolia Uline ex R. Knuth, Notizbl. Königl. Bot. Gart. Berlin 7(65): 186. 1917.— TYPE: BRAZIL. Minas Gerais: Itacolomy, 24 Jan 1893, C. A. W. Schwacke 9032 (holotype: B!). + Dioscorea campestris f. piedadensis Uline ex R. Knuth, Notizbl. Königl. Bot. Gart. Berlin 7(65): 186. 1917.—TYPE: BRAZIL. Minas Gerais: in Serra de Piedade, 1893, J. E. B. Warming s. n. (lectotype: C10010728!, here designated). + Dioscorea campestris var. longispicata Hauman, Anales Mus. Nac. Hist. Nat. Buenos Aires 27: 451. 1916.—TYPE: ARGENTINA. Prov. de Misiones: Posadas, 15 Jan 1907, C. L. Spegazzini 20711 (lectotype: BR!, here designated). herbaceous, 2 mm diam. at base; cataphylls present towards stem base, rather brittle, lanceolate, lateral nodal flanges and bulbils not present, 0.5–1 mm diam. on apical shoots. Leaves alternate, entire, monomorphous; petiole 0.4–2 cm long, with short basal and apical pulvini, twisted at base, flat to slightly canaliculate, glabrous; blade 5–12 1.2–5 cm, dark green above and bright green below, glabrous on both sides, chartaceous, ovate to narrowly elliptic, with very narrow sinus 1–4 mm deep, base cordate with rounded lobes, apex acuminate, bearing an acuminate forerunner tip 0.8–2.1 mm long, protruding veins 3–7 below. Staminate inflorescence 6.5–25 cm long, initially erect, becoming pendent, one per axil, heterothetic compound inflorescence with racemes on principal axis and drepanium on secondary axis, 1–7 flowers per rachis node, bracts present at pedicel base and at drepanium base and branching points. Stami0.3–0.6 mm, nate flowers pedicellate, floral bracts 0.8–1.3 ovate to lanceolate, membranous, torus deeply convex, perianth light green, slightly campanulate, lobes reflexed, inner 1–1.4 mm, oblong to obovate, and outer tepals 1.5–2 glabrous, with fine midrib; stamens six, inserted at base of perianth segments, exserted, filaments ca. 2 mm long, free, anthers ca. 0.3 mm long, pistillodes inconspicuous (Figs. 3A– D; 4D). Pistillate inflorescence 2 –8.6 cm long, simple, spicate, pendent, one per axil, 1 or 2 bracts at pedicel base. Pistillate flowers sessile, one per rachis 0.3–0.6 mm, oblong to ovate – node, floral bracts 1.5–2 acuminate, torus shallowly convex, perianth yellowish to light brown, campanulate, internal and external tepals 1–1.5 0.6–0.8 mm, ovate–acuminate, glabrous, with fine midrib; gynoecium tricarpellate, styles 3, fused to form a 1 mm long, stout, erect, cylindrical column, 3-lobed at apex, each lobe recurved and splitting into two, stigmatic surfaces flat; staminodes six, ca. 1 mm long, antheriform; ovary dark green, glabrous (Figs. 3F–I; 4E). Capsules 0.8–1 cm, dark brown to whitish, oblong to 1.6–1.9 elliptic, initially pendent, reflexing during maturation to erect positions, valves chartaceous when mature, opening + “Habitat in provinviae Minarum Generalium campis editis, ubi inter herbas scandit”, s. d., C. F. P. Martius 40 (lectotype: M!; isolectotype: K, here designated). Brazil, restricted to fields at altitudes of 2,300–2,790 masl Medium size (27.2 mm) Monocolpate with long and wide colpi (ca. 23.1 7.2 mm) Columellae with low muri and sparsely perforate, absent or very few granules within the lumen; smaller lumina around the larger lumina 2–2.5 15–35 3.3–6.5 Subcoriaceous Ovate, spreading Rhizomatous appearance, with many meristematic points from where the aerial stems emerge Annual Elliptical, papyraceous, with smooth margin Wide and parenchymatous Two vascular bundles with only one vessel of larger diameter and the others with two Brachysclereids absent + Table 4. COUTO ET AL.: REVISION OF DIOSCOREA CAMPESTRIS 1060 SYSTEMATIC BOTANY [Volume 39 Fig. 2. A–D. Cross section showing anatomy of aerial stem and tuber. A and C. Dioscorea campestris. B and D. Dioscorea pedalis. A–B. Aerial stem. Note difference in composition and size of medulla between species. White circles indicate cauline vascular bundles and black circles identify common bundles; white arrows (phloem unit); lv (large vessel); pi (pith). C–D. Tuber. Note brachysclereids (asterisk) in storied cork (sto). Scale bars: A = 150 mm; B, C = 150 mm; D = 400 mm. Palynology—Small (20.5 mm) (Tables 2, 3), heteropolar (Fig. 1A), in monads, amb elliptic, monocolpate with long and wide colpi (ca. 17.2 4.0 mm) with thick edges (Fig. 1A), psilate. Ornamentation reticulate, heterobrochate (Fig. 1A – B); columellae simple, with low muri and sparse perforations (Fig. 1A – B), with granules within the lumen and lumina of generally uniform sizes (Fig. 1B). Sexine (ca. 1.0 mm) almost as thick as the nexine (ca. 0.9 mm). Distribution and Ecological Data—Distributed in Argentina, Brazil, and Paraguay, being found from Amazonas State in Brazil to Buenos Aires Province in Argentina (Fig. 5). This species inhabits dry to fairly moist locations, such as rocky outcrops, the edges of evergreen rainforests, grasslands, gallery forests, and rocky fields at lower altitudes (maximum of 1,800 m above sea level (masl)). + + from ca. ⅓ to ⅔ of their length, glabrous, perianth traces at apex, margin dilated; seeds 0.6–0.8 cm long, dark brown, oblong, wing 1.3–1.6 0.6–0.9 mm, elongated to seed base (Figs. 3J –K; 4F). Anatomy—The aerial stem has a uniseriate epidermis with a thick cuticle. Angular collenchyma constitutes about three cell layers, while the rest of the cortex is composed of parenchyma cells. An endodermoid layer has parenchyma cells with lignified walls. The central cylinder has a peripheral fibrous ring. The vasculature consists of three common vascular bundles and three cauline vascular bundles (Fig. 2A). Both types of vascular bundles have one pair of large vessels and two to three phloem units; the pith is extremely reduced and strongly sclerified (Fig. 2A). Storied cork with approximately three layers of brachysclereids covers the tuber (Fig. 2C). 2014] COUTO ET AL.: REVISION OF DIOSCOREA CAMPESTRIS 1061 Fig. 3. Dioscorea campestris. A. Staminate inflorescence. B. Secondary staminate inflorescence. C–D. Details of staminate flower. E. Habit. F. Pistillate inflorescence. G. Secondary pistillate inflorescence. H–I. Details of pistillate flower. J. Infructescence. K. Fruit. L. Tuber in apical view. M. Tuber with branch in side view. Scale bar: A = 5 cm; B, C, H, I = 3 mm; D = 1.5 mm; E = 4 cm; F = 1 cm; G = 4 mm; J = 3.5 cm; K = 2.2 cm; L, M = 14 cm. 1062 SYSTEMATIC BOTANY [Volume 39 Fig. 4. Dioscorea campestris. A. Habit. B. Tuber in side view. C. Tuber in apical view. D. Detail of staminate flower. E. Detail of pistillate flower. F. Fruit. Scale bars: A = 2.5 cm; B = 3.5 cm; C = 4.5 cm; D = 1 mm; E = 1.5 mm; F = 1 cm. Variations could be observed in the sizes of the leaves and inflorescences, often related to the ages of the individuals, ambient humidity, or insolation. Large variations in the shapes of the leaves are uncommon, but when these occur they are usually associated with the positions of the leaves on the stem (apex–base). In the case of the Dioscorea campestris assemblage, variations observed in leaves and inflorescences were not the main motivating factor for describing the taxa treated here as synonyms, as can be seen in the treatment by Knuth (1924). Conservation Status—Least concern (LC), as the species is widely distributed, with a continuous area of occupation. It is found in several legally protected areas in Argentina, Brazil, and Paraguay. 2014] COUTO ET AL.: REVISION OF DIOSCOREA CAMPESTRIS 1063 Fig. 5. Distribution map of Dioscorea campestris and Dioscorea pedalis based on herbarium specimens. Phenological Data—Plants flowering from November to March, with fruits being present from November to April. Etymology—The specific epithet comes from the Latin “campestris”, meaning from the field, in reference to the type locality in the high altitude fields of Minas Gerais State, Brazil. Representative Specimens Examined—ARGENTINA. Corrientes: Dept. Santo Tomé, Potrero Luna (28
210 S, 55
470 W), 5 Dec 1981, S. G. Tressens et al. 1630 (CTES); Missiones: Missiones, Dep. Iguazú, Ruta 101, 11 Jan 1972, A. Nandú & L. Mrogiaski 315 (RB); Dept. Concepción, Ruta 2, 16 km NE de Azara, 4 Feb 1982, A. Schinini et al. 21841 (CTES); Salta: Rivadavbia, Los Blancos, Barrio Mataco (23
360 S, 62
350 W), 24 Jan 1983, A. Maranta & P. Arenas 177 (NY). BRAZIL. Alagoas: Pão de Açúcar, Mata da Onça, (9
440 200 S, 37
340 3500 W), 04 May 2002, R. P. Lyra-Lemos et al. 6673 (HUEFS). Bahia: Bom Jesus da Lapa, rio São Francisco, 15 Apr 1980, R. Harley 21369 (K, RB). Ceará: Jaguaretama, 03 May 1984, M. C. Pinheiro 153 (R). Distrito Federal: Brası́lia, 21 Aug 1973, E. P. Heringer 12869a (HB). Minas Gerais: Serra da Grão Mongol, 12 Nov 1938, F. Markgrat et al. 3476 (RB); Lima Duarte, Parque Estadual do Ibitipoca, 23 Jan 2007, R. C. Forzza 4384 (RB). Paraná: Guarapuava, Canta Galo, 01 Feb 1985, G. Hatschbach & A. C. Cervi 48877 (PEL). Pernambuco: Custódia, Reservatório Bagres, Lote 10 (08
200 06.0000 S, 037
460 58.8000 W), 14 Ago 2011, R. S. Couto 531 (HVASF); Alagoa de baixo, 30 Mar 1991, H. Monteiro 350 (RBR). Rio de Janeiro: Itatiaia, Macieiras, 26 Feb 1945, A. C. Brade 17508 (RB); Itatiaia, Lote 28–30, 5 Feb 1948, A. C. Brade 18821 (COL, RB); Teresópolis, Parque Nacional da Serra dos Órgãos, trilha para a Pedra do Sino, 31 Mar 2010, R. S. Couto et al. 315 (R, RB, RFA); São Fidelis, estrada para Ayres sentido Santa Maria Madalena, 28 Ago 2009, R. S. Couto et al. 232 (R, RB, RFA); São Fidelis, estrada para Ayres sentido Santa Maria Madalena, 28 Ago 2009, R. S. Couto et al. 233 (RB, RFA); Parque Nacional do Itatiaia, trilha para a Cachoeira Poranga, 20 Jul 2010, R. S. Couto 375 (RB, RFA). Rio Grande do Sul: Vila Olivia para Caxias, 28 Jul 1954, B. Rambo s. n. (PACA 31292); General Câmara, Santo Amaro, 08 Jan 1978, J. L. Waechter 703 (ICN). Santa Catarina: Lages, 25 Dec 1956, J. Mattos s. n. (PACA 61063). São Paulo: Fernandópolis, Mata do Zoológico, 10 Jun 1993, R. N. Damasceno 223 (RB, RUSU); Salesópolis, 22 Nov 1957, M. Kuhlmann 4287 (SPF). PARAGUAY. Alto Paraguay: Mayor Pablo Lagerenza, 16 Apr 1978, A. Schinini & E. Bordas 14986 (CTES); Paraguay: Cerro Santo Tomás, Dec 1971, A. Schinini 4212 (CTES). 1064 SYSTEMATIC BOTANY Notes—Dioscorea campestris, with two varieties, was described by Grisebach (1842) based on Martius 40 (M) and 39 (M), and Sellow 50 (B, K, and P), all of which were collected in Minas Gerais State. The materials cited by Grisebach (1842) have no collection numbers (like most other Dioscorea species described by him), but by examining the types deposited in B, M, and P it was possible to encounter labels with collection number designations in the handwriting of the collectors (Sellow and Martius), as well as the identification of D. campestris in the handwriting of Grisebach. Pedralli (2002, 2004) proposed the lectotypification of Martius s. n. (M175806). Upon close examination, we found that all of the syntypes perfectly matched the description of Grisebach (1842) for D. campestris. Dioscorea campestris var. grandiflora and D. campestris var. parviflora (Grisebach 1842) were described without any indication of the holotype material. Pedralli (2004) defined them as “nomen nudum”, and justified this proposal based on the lack of indication of the type materials. The present work accepts them as validly published based on Art. 37.1 (McNeill et al. 2012), and we here propose lectotypification of the collection Sellow 50 (K) for Dioscorea campestris var. grandiflora and the collection Martius 40 (M) for D. campestris var. parviflora, as they best represent the characteristics described by Grisebach (1842), and are better preserved. However, we do question the morphological criteria presented by Grisebach (1842) for distinguishing the varieties. We observed substantial morphological variability in the vegetative and sexual organs of D. campestris, but without any obvious patterns that would allow the recognition of infraspecific taxa, especially those designated by Grisebach (1842) for these two varieties (based on flower size and pedicel length). Therefore, these two varieties are considered here as synonyms of D. campestris. The new combination Helmia campestris was proposed by Kunth (1850) based on D. campestris (Grisebach 1842). We consider the genus Helmia to be artificial and not worthy of recognition, as noted previously by Grisebach (1875), Uline (1897), Knuth (1917, 1924), Burkill (1960), Waitt (1963), Barroso et al. (1974), Xifreda (1989), Al–Shehbaz and Schubert (1989), and Caddick et al. (2002), among others. In their phylogenetic study, Wilkin et al. (2005) likewise demonstrated that the morphological seed characters on which Helmia was based (seed wing elongate to base) had multiple origins within Dioscorea, indicating this genus as paraphyletic. Dioscorea campestris var. longispicata was described by Hauman (1916) based on Spegazzini 20711 (BR) and 18564 (BR), and Rodriguez 325 (BR). All three specimens were collected in the Misiones Province in Argentina, but without any indication of the holotype. Pedralli (1998) indicated Spegazzini 20711 as the lectotype in his thesis, but this was not a valid publication. Pedralli (2004) later proposed subsuming this variety within D. campestris, but without validating the lectotypification. We have chosen Spegazzini 20711 as the lectotype, as it best represents the characteristics described by Hauman (1916) and is well-preserved. Dioscorea campestris f. plantaginifolia was described by Knuth (1917) based on Schwacke 9032 (B) collected in Itacolomi, Brazil, and on Ule 3763 (B) from Itatiaia, Brazil. Subsequently, a new description for the form was presented by Knuth (1924), together with morphological data indicating the holotype as Schwacke 9032 (B). After [Volume 39 analyzing the types, we determined that D. campestris f. plantaginifolia shows no significant morphological differences from D. campestris. Dioscorea campestris f. stenorachis was described by Knuth (1917) based on Glaziou 14351, collected in Petrópolis, Brazil. Knuth (1924) added the collection Miers 4149 to the examined material and designated Glaziou 14351 (deposited in B) as the holotype. This material could not be found in B, however, probably having been destroyed in 1943. Pedralli (1998) wrongly designated Miers 4149 as the holotype. After analyzing Glaziou 14351 (C, G, and P) it was determined that D. campestris f. stenorachis is a synonym of D. campestris, as the main feature used by Knuth (1917) to describe the new form (staminate inflorescences two times longer than D. campestris) can be observed in Glaziou 14351 (C, G, and P). Additionally, the D. campestris material examined had staminate inflorescences ranging from 6.5 cm (as described for D. campestris by Grisebach [1842]) to 25 cm (as described by Knuth [1924] for D. campestris f. stenorachis). After analyzing the isotypes (C, G, and P), we here designate the collection Glaziou 14351, deposited in P (P00748327), as the lectotype as it perfectly represents the characters described by Knuth (1917) and is in better condition than the others. Dioscorea campestris f. piedadensis was described by Knuth (1917) based on Warming s. n. collected in Serra de Piedade, Brazil. Knuth (1924) subsequently indicated Warming s. n. (deposited in B) as the holotype in his revision of Dioscoreaceae. However, this material is not in the Berlin herbarium (B) and was probably destroyed during the bombings of 1943. In examining the isotype (C), it was concluded that no morphological differences exist when compared to D. campestris and, due to the destruction/disappearance of the holotype, we here designate Warming s. n. (C10010728) as the lectotype. Knuth (1926) proposed Dioscorea campestris f. paraguayensis based on Anisits s. n. (B 100250036) from Paraguay. The main argument used by this author to establish the new form was the morphological differences of its leaves. However, examination of the holotype (B) showed that its floral traits coincided with those of D. campestris and that the differences observed between their leaves are consistent with existing morphological variation in this species. Dioscorea campestris Kunth was a synonym proposed by Pedralli (2004) for the species D. campestris. Pedralli (2004) justified this proposal by claiming that this taxon was a superfluous name according to Article 52 (McNeill et al. 2012). However, when considering the publication of Kunth (1850), only Helmia campestris (Griseb.) Kunth was described, a validly published combination. The name D. campestris Kunth was not mentioned anywhere by Kunth (1850). Therefore, the proposal of Pedralli (2004) cannot be considered, and D. campestris Kunth is a “nomen nudum”. Dioscorea pedalis (Uline ex R. Knuth) R. Couto and J. M. A. Braga, comb. et stat. nov. Dioscorea campestris f. pedalis Uline ex R. Knuth, Notizbl. Königl. Bot. Gart. Berlin 7(65): 187. 1917.—TYPE: BRAZIL. Rio de Janeiro: Itatiaia, Fazenda do Rio Preto, en haut de la Serra près des campos, 21 Jul 1876, A. F. M. Glaziou 8993 (holotype: B!; isotype: P!) Left-twining vine, annual, dioecious (Figs. 6A; 7A). Tuber with a rhizomatous aspect, with many meristematic points 2014] COUTO ET AL.: REVISION OF DIOSCOREA CAMPESTRIS 1065 Fig. 6. Dioscorea pedalis. A. Habit. B. Secondary staminate inflorescence. C–D. Details of staminate flower. E. Staminate buds. F. Pistillate inflorescence. G–H. Details of pistillate flower. I. Fruit. J. Tuber with branch in side view. K. Cross section of tuber. Scale bars: A = 2 cm; B = 5 mm; C, H = 3 mm; D = 4.5 mm; E = 2 cm; F, K = 1.5 cm; G = 3.5 mm; I = 1 cm; J = 5 cm. 1066 SYSTEMATIC BOTANY [Volume 39 Fig. 7. Dioscorea pedalis. A. Habit. B. Tuber with branch in side view. C. Staminate inflorescence. D. Cross section of tuber. E–F. Details of pistillate flowers. G. Fruit. Scale bars: A = 5 cm; B = 1.5 cm; C = 1.5 mm; D = 2.5 mm; E = 1.2 mm; F = 1 mm; G = 5 mm. to deltoid, lateral nodal flanges and bulbils not present; in apical shoots ca. 2 mm in diam. Leaves alternate, entire, monomorphous; petiole 0.5–1 cm long, with short basal and apical pulvini, twisted at base, strongly canaliculate, glabrous; blade 2.8–5.2 1.3–2.8 cm, dark green above and bright green below, glabrous on both sides, subcoriaceous, ovate to triangular, with narrow sinus 3–11 mm deep, base + from which aerial stems grow, with fine roots emerging at many different points along the tuber, periderm dark brown to black, parenchyma yellow, ca. 5 cm below soil surface (Figs. 6J–K; 7B; 7D). Stems 15–35 cm long, initially erect to twining, glabrous, cylindrical, striated, unarmed, green, herbaceous, 2.5 mm in diam. at base; cataphylls present only at first node, rather brittle, shortly lanceolate 2014] COUTO ET AL.: REVISION OF DIOSCOREA CAMPESTRIS + + + + + + + cordate with rounded lobes, apex attenuated, bearing an acuminate forerunner tip 1.1–3 mm long, protruding veins 5–9 below. Staminate inflorescence 3.3–6.5 cm long, erect to patent, 1–3 per axil, heterothetic compound inflorescence with racemes on the principal axis and drepanium on the secondary axis, 1–5 flowers per rachis node, bracts present at pedicel base, drepanium base, and at branching points. Staminate flowers pedicellate, floral bracts 1–1.5 0.3–0.5 mm, ovate to oblong–acuminate, membranous, torus deeply convex, perianth greenish white to yellowish, campanulate, lobes patent, inner and outer tepals 2–2.5 1.3–1.8 mm, ovate, glabrous, with fine midrib; stamens six, inserted at base of perianth segments, exserted, filaments ca. 2 mm long, free, anthers ca. 0.5 mm long, pistillodes central, 3–lobed at apex (Figs. 6B –E; 7C). Pistillate inflorescence 1.2–3.4 cm long, simple, spicate, patent, one per stem node, 1 or 2 bracts at pedicel base. Pistillate flowers sessile, one per rachis node, floral bracts 2–2.5 0.8–1.1 mm, ovate to ovate–acuminate, torus deeply convex, perianth yellowish white to greenish white, campanulate, internal and external tepals 2 1.4–1.5 mm, ovate, glabrous, with fine midrib; gynoecium tricarpellate, styles 3, fused to form a 1.3 mm long, stout, erect, cylindrical column, 3-lobed at apex, each lobe recurved and split into two rounded stigmatic surfaces; staminodes six, ca. 1 mm long, antheriform; ovary dark green, glabrous (Figs. 6F–H; 7E–F). Capsules 0.8–1.2 0.4–0.8 cm, dark brown to light brown, elliptic, initially pendent, reflexing during maturation to erect positions, valves papyraceous when mature, opening to ca. ⅓ of their length, glabrous, perianth traces at apex, margin not dilated; seeds 0.5–0.9 cm long, light brown, elliptic, wing 0.7–1.1 0.3–0.6 cm, elongated toward seed base (Figs. 6I; 7G). Anatomy—The stem has a uniseriate epidermis with a thick cuticle. Angular collenchyma is about three layers thick, with the rest of the cortex consisting of parenchyma cells. The endodermoid layer has parenchyma cells with lignified walls. The central cylinder has a peripheral fibrous ring. The vasculature consists of four common vascular bundles and five cauline vascular bundles. Both vascular bundle types have one pair of large vessels and two to three phloem units. The central cylinder contains two peripheral vascular bundles with only one large vessel each. Fibers fill all vascular bundles, while the pith is wide and composed of parenchyma cells with thin walls. The storied cork, which does not contain brachysclereids, serves as the tuber covering. Palynology—Medium-sized (27.2 mm) (Tables 1, 2), heteropolar (Fig. 1D), in monads, amb elliptic, monocolpate with long and wide colpi (ca. 23.1 7.2 mm) with thick edges (Fig. 1D), psilate. Ornamentation reticulate, heterobrochate (Figs. 1D–F); columellae simple, with low muri and sparse perforations (Figs. 1D–F), granules absent (or very few) within the lumina and smaller lumina around the larger lumina (Fig. 1F). The sexine (ca. 1.0 mm) is almost as thick as the nexine (ca. 0.9 mm). Distribution and Ecological Data—Dioscorea pedalis is restricted to high altitude fields (2,300–2,790 masl), only inside Itatiaia National Park (PNI) in Rio de Janeiro State, Brazil (Fig. 5). The species inhabits cracks in the rocks that have accumulated substrate and have higher humidity levels, preferably shaded hollows in the rock where solar luminosity is less intense. 1067 This species does not display major morphological variations; leaf shape, size, and consistency are fairly uniform, as are those of the floral parts. Conservation Status—Critically endangered (CR). This species is rare, occurring only in high altitude grasslands, and is restricted to an area of less than 10 km2 in the Itatiaia National Park. Even though this species occurs in a protected area, various threats to biodiversity still exist in the PNI, including hunting, illegal extraction, unsupervised tourism, unregulated land use, and (especially) burning (Aximoff and Rodrigues 2011), putting the conservation of this species at further risk. Phenological Data—Plants flower and fruit from October to January. Etymology—The epithet derives from the Latin “pedalis”, meaning measuring a foot, referring to the size of the plant. Representative Specimens Examined—BRAZIL. Rio de Janeiro: Parque Nacional do Itatiaia, Prateleiras, 18 Jan 1935, P. Campos Porto 2842 (RB); Parque Nacional do Itatiaia, Planalto, 20 Oct 2009, R. S. Couto & L. J. T. Cardoso 265, 266, and 267 (RB, RFA); Parque Nacional do Itatiaia, ca. 50 m do Abrigo Rebouças, 07 Nov 2011, R. S. Couto et al. 587 (RFA); Parque Nacional do Itatiaia, base das Prateleiras, 07 Nov 2011, R. S. Couto et al. 593 (RB); Parque Nacional do Itatiaia, Abrigo Rebouças, alt. 2300m, 11 Oct 1977, P. J. M. Maas & G. Martinelli 3183 (RB); Itatiaia, base das Agulhas, 28 Nov 1938, F. Markgraf & A. C. Brade 3712 (RB); Itatiaia, 2400m, 27 Nov 1952, F. Markgraf & A. C. Brade 21262 (RB); Parque Nacional do Itatiaia, Prateleiras, 28 Dec 1934, R. K. F. Pilger & A. C. Brade 49 (RB). Notes—Dioscorea campestris f. pedalis was described by Knuth (1917) based on Ule 4065 (B) and Glaziou 8993 (B), both from the region now known as Itatiaia National Park. Subsequently, Knuth (1924) clearly designated the holotype as Glaziou 8993 (B), and mentioned that both materials of the original publication can be found in Berlin. Analysis of Ule 4065 (B) and Glaziou 8993 (B and P) indicated that their morphological characteristics coincided with those presented by Knuth (1917, 1924), although D. campestris and D. campestris f. pedalis do differ in several respects, including anatomical and pollen characteristics. Adding to the information obtained in the field, we propose that D. campestris f. pedalis be treated as a species distinct from D. campestris, as described below. Discussion Dioscorea campestris and D. pedalis have monosulcate pollen. This is the most common aperture type among monocots (Penet et al. 2005), as seen in recent work with the Asparagaceae (Lopes et al. 2013). They both show reticulate sexine, although great ornamentation diversity can be found in the family (Caddick et al. 1998; Wilkin and Caddick 2000; Schols et al. 2001; Schols et al. 2003; Schols et al. 2005; Wilkin et al. 2009). Overall, however, the Dioscorea species analyzed here can be separated based on the sizes of their pollen grains and sexine ornamentation; D. pedalis has smaller lumina around the larger lumina, and granules are absent (or very few) within the lumina, while in D. campestris the lumina are more uniform in size and granules are present within the lumina. Anatomical characters have historically been useful for resolving taxonomic problems. Ayensu (1972) reported that the pith in Dioscoreaceae shows significant variation, which can be taxonomically useful at the species level. Dioscorea pedalis has a voluminous and parenchymatous pith, whereas it is reduced and sclerified in D. campestris. 1068 SYSTEMATIC BOTANY Both species form storied cork, although only D. campestris has brachysclereid layers in this tissue. According to Haberlandt (1928), brachysclereids are typical of woody eudicotyledons and increase the incompressibility of the bark, although Tenorio et al. (2012) observed these cells in the storied cork of Philodendron Schott species. Overall, significant differences in stem anatomy and pollen morphology between these two taxa support the recognition of D. campestris f. pedalis at the species level, requiring the new combination D. pedalis. Acknowledgments. The authors thank Rachel Dana for the drawings of both species for this article; Leandro Jorge Telles Cardoso and Luana Mauad for their company in the field; Roy R. Funch for the English revision; Allan J. Bornstein for the valuable comments that improved our manuscript; and CAPES and CNPq for their financial support through grants to the first and second authors. Literature Cited Al–Shehbaz, I. A. and B. G. Schubert. 1989. The Dioscoreaceae in the southeastern United States. Journal of the Arnold Arboretum 70: 57–95. Aximoff, I. and R. C. Rodrigues. 2011. Histórico dos incêndios florestais no Parque Nacional do Itatiaia. Ciência Florestal 21: 83–92. Ayensu, E. S. 1972. Dioscoreales. 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