A revision and morphological analysis of the Uruguayan species of Stevia (Compositae, Eupatorieae)

A revision and a morphological analysis of the Uruguayan species of Stevia (Compositae, Eupatorieae) were performed. Leaf, inflorescence, pubescence and pappus traits were identified as key to separate species. Stevia entreriensis , S. entreriensis var. minor , and Dissothrix hassleriana were considered synonyms of S. hirsuta , and S. ophryodonta and S. oxylaena synonyms of S. veronicae . Lectotypes for the names Stevia cinerascens , S. megapotamica , S. linariifolia , S. selloi , S. selloi var . yparacayensis , S. oxylaena and S. veronicae were designated. Stevia burkartii was excluded from the Uruguayan flora. As a result, 10 Uruguayan species are considered: S. aristata , S. cinerascens , S. congesta , S. gratioloides , S. hirsuta , S. multiaristata , S. sabulonis , S. satureiifolia , S. selloi , and S. veronicae . A key to the Uruguayan species, descriptions, photographs and distribution maps are provided.


Introduction
Asteraceae is one of the largest vascular plant families with 25,000-33,000 species (Anderberg et al. 2007;Robinson et al. 2009;Mandel et al. 2017) and has a high number of genera and species particularly in the Americas, and especially in South America where its origin has been postulated (Barreda et al. 2010(Barreda et al. , 2012. The genus Stevia Cav. (Cavanilles 1797) stands out within Asteraceae for its wide distribution, from southern United States up to northern Chile and northern Patagonia in Argentina, inhabiting different type of environments (King & Robinson 1987;Freire & Ariza Espinar 2014;Gutiérrez et al. 2016), and high number of taxa with ca. 175-237 species (King & Robinson 1987; Rodriguésia 70: e01532018. 2019 2007; Robinson et al. 2009), with high speciesdiverse areas in Mexico (Watanabe et al. 2001;Turner 2015) along with South America (e.g., Freire & Ariza Espinar 2014;Robinson 2014). It includes the South American species S. rebaudiana (Bertoni) Bertoni, commonly known as candyleaf, with food, industrial and medicinal uses (Brandle et al. 1998;Soejarto 2002;Salvador-Reyes et al. 2014). This genus belongs to the subtribe Piqueriinae of the tribe Eupatorieae, a derivate evolutionary lineage of Asteraceae (Robinson et al. 2009;Tippery et al. 2014;Rivera et al. 2016).
Stevia is a morphologically well circumscribed genus and comprises herbs or shrubs with opposite leaves, cylindrical involucre of five phyllaries, five florets per capitulum with funnelform corollas, and 5-ribbed, pubescent cypselae. The pappus, generally constituted by bristles, short scales, or both, has been traditionally used for species or infrageneric delimitations (Robinson 1930;Bremer et al.1994;. At species level, the morphology turns out to be complex since there is overlapping of characters and, in most cases, species delimitations are not clear. Infrageneric classifications of Stevia have been made based especially on features of the habit, inflorescence, fruit, and pappus. Baker (1876) proposed the serie categories Paleaceoaristatae, Pauciaristatae and Multiaristatae for Brazilian and bordering countries species, while Robinson (1930) established the series Corymbosae and Podocephalae for Central and North America. Currently, there is no agreement for recognizing these infrageneric categories , although some of them have been used for a comprehensive approach to the Brazilian species (Monteiro 1982;Nakajima 1991). In addition, molecular phylogenetic evidence indicated that these categories are not monophyletic (Soejima et al. 2017).
In summary, Stevia is a complex genus and a major taxonomic challenge due to its high number of species, wide geographic distribution, and species variability of morphological characters. One way of untangling the taxonomy of the genus is its treatment by biogeographical areas or by countries. In southern South America, some taxonomical advances have been achieved through checklists (e.g., Freire 2008;Nakajima 2010;Robinson 2014;Avila et al. 2016), floristic treatments (e.g., Cabrera 1996;Freire & Ariza Espinar 2014) and revisionary studies (Robinson 1930;Gutiérrez et al. 2016). Uruguay is one of the South American countries that still lacks a taxonomical treatment of Stevia although Asteraceae is represented by 374 species and constitutes the second family of vascular plants in species number (Zuloaga et al. 2008).
The taxonomical history of Stevia in Uruguay started within the genus Eupatorium; Lamarck (1786) described Eupatorium satureiifolium, currently Stevia satureiifolia (Lam.) Sch. Bip. ex Klotzsch (1852), on the basis of a specimen from Montevideo. The generic name Stevia was created by Cavanilles (1797) who established three Mexican species. In the following years, six Uruguayan species were described or transferred from other genera: S. multiaristata Sprengel (1826) Kuntze (1898).
As a part of an ongoing revisionary study of the southern South American species of Stevia, the goal of this work is to clarify and update the taxonomy of the Uruguayan species by means a revision and a deep morphological analysis of the genus Stevia in Uruguay.

Material and Methods
For this study 300 specimens were examined from BA, BAB, BAF, CORD, CTES, LIL, LP, MVFA, MVJB, MVM and SI (Thiers, continuously updated). Additional specimens were examined as digital images available through online resources (BM, E, F, G, GH, GOET, K, NY, P, S, US, Z and other online resources, such as the JSTOR Global Plants web site).
Descriptions are based on herbarium specimens and field observations. Several field trips were made throughout Uruguay and vouchers were deposited in BA, LP and/or MVFA. A l l s p e c i e s w e r e m a c r o -a n d micromorphologically analyzed in detail, especially those previously used for delimiting species (i.e., leaf, trichomes, and pappus traits); in all, 57 specimens were measured. The five florets of one to four capitula per specimen, all at the same stage of maturity, were analyzed. For light microscope examination, leaves were rehydrated, treated with a clearing process (Zarlavsky 2014) and stained with 2% safranin. In addition, light microscope observations and photographs were taken on a Nikon Eclipse E200 microscope equipped with a digital camera. For scanning electron microscopy (SEM) studies, dry material was placed directly on the stubs and coated with palladium/gold. The samples were scanned and photographed in a Philips XL-30 SEM (Museo Argentino de Ciencias Naturales).
Drawings of Stevia veronicae were made by J.F. Rodríguez-Cravero and D.G. Gutiérrez using a Wild Heerbrugg M3 stereomicroscope with a camera-lucida attachment, and inked by V. Piñón.

Results
According to our results, we recognize 10 species of Stevia instead of 11 previously cited for Uruguay (i.e., Freire 2008;Rodríguez-Cravero et al. 2017). These ten species of Stevia are present in Uruguay: S. aristata, S. cinerascens, S. congesta, S. gratioloides, S. hirsuta, S. multiaristata, S. sabulonis, S. satureiifolia, S. selloi and S. veronicae. All the Uruguayan species of Stevia would belong to the series Multiaristatae (Candolle 1836; Grasshof et al. 1972) for showing more than 14 bristles per cypsela (at least in the adelphocarps), that are as long as, or somewhat longer than, the corolla.
These species are recognized by key features of stem branching, length of the internodes, the shape, size and arrangement of the leaves, general shape of the inflorescence, length of the peduncles, number of heads, color of the flowers, pappus, bristle size and pubescence of the plant. With the exception of the trichomes, these characters were previously employed by other authors to distinguish species. Variation in the type and number of hairs in phyllaries and peduncles was observed among individuals of the same species, and therefore have not taxonomic relevance.

Phyllotaxy and leaf traits
There are three main types of leaves arrangement: (1) the leaves of the main stem are clustered at the nodes, apparently verticillate (actually opposite) because the internodes are very short; with internodes of the lateral branches also very short (Fig. 1a); (2) the leaves of the main stem are conspicuously opposite and sparsely distributed with long internodes; in this case, the leaves of the lateral branches are apparently verticillate (Fig.  1b) as type one; and (3) leaves of the main stem and that of the lateral branches are conspicuously opposite (sometimes alternate in the upper leaves) and sparsely distributed with long internodes (Fig.  1c). There are intermediate leaf arrangement types.
In general, bigger leaves are found in mature individuals, located at the base of the plant and disposed on the main stem. Leaves are usually smaller in the upper part of the plant (e.g., S. aristata, S. cinerascens) but sometimes they are of the same size throughout the whole stem (e.g., S. satureiifolia, S. hirsuta).
Two main types of leaves were identified: (1) Linear type: sessile leaf with linear blade, up to 5 cm long and 7(-8) mm wide, sometimes fleshy with entire margin (S. satureiifolia, Fig. 2a) or not-fleshy and denticulate (S. multiaristata), a conspicuous midvein, and glandular pubescence, associated with an apparently verticillate phyllotaxy (see above type of phyllotaxy). This combination is found in S. multiaristata and S. satureiifolia; (2) Non-linear type: Sessile or pseudopetiolate (i.e., with an inconspicuous short winged petiole) leaves Figure 1 -a-c. Phyllotaxy and leaf traits in Uruguayan Stevia -a. leaves densely grouped in the main stem as in S. satureiifolia; b. leaves densely grouped in secondary branches as in S. congesta; c. evenly spaced leaves with opposite arrange in the primary stem as in S. aristata. with narrowly elliptic, elliptic, ovate, obovate or rhomboidal blade, up to 9 cm long, up to 6 cm wide, entire, crenate or serrate margins, 1-or 3-nerved, and glandular to hirsute pubescence, associated with a conspicuously opposite phyllotaxy. Stevia aristata, S. cinerascens, S. congesta, S. gratioloides, S. hirsuta (Fig. 2), S. sabulonis, S. selloi, and S. veronicae show this combination. The blade apex is generally acute, but it is obtuse in S. congesta, S. sabulonis, and S. veronicae. The blade base may be rounded, cuneate, or shortly decurrent (pseudopetiolate).

Inflorescences
The heads of the species of Stevia are sessile or pedunculate (up to 2 cm long); if the peduncle is longer than the height of the involucre (ca. 6 mm), it is considered here as a long pedunculate capitulum. The length of the capitula peduncles generally determines the type of secondary inflorescences as following.
The capitula of Stevia are borne in two main types of cymose inflorescences: (1) a corymbiform cyme, which has shortly pedunculate capitula, clustered at the top of the lateral branches, densely arranged (Fig. 3a). This type of cyme shows two variations; in one case, lateral and terminal flowering branches reach the upper part of the inflorescence (it is commonly named as "paucicephalous"; e.g. S. hirsuta), or, in the other case, the lateral flowering branches are shorter than main flowering axis (i.e. "pluricephalous"; e.g. S. aristata); and (2) a paniculiform cyme with few or solitary long pedunculate capitula at the top of the lateral branches, laxly arranged (Fig. 3b).
Head, when present, 4-5-celled in each row, of varied shapes, rounded or emarginate. Cuticular vesicle enclosing the last tier of cells or restricted to the apex, persistent or collapsing early. Found in stems, leaves, peduncles, phyllaries and externally in corollas.
(3) Cylindrical: foot 1-celled, body cylindrical, entire, uniseriate, 2-6-celled, cells usually longer above, occasionally the basal ones broader than long, cross walls slightly demarcated, cross and lateral walls thin, acute at the apex, smooth or slightly ridged (Figs. 4d; 5e). Found in stems, leaves ( Fig. 2c), peduncles, phyllaries, and externally and internally in corollas. It is important to note that in Stevia this type of hair appears in an annular arrangement at the base of the lobes and the upper part of the throat inside the corolla.
(4) Twin: usually short (approximately 30 μm long), two short basal cells (one sometimes reduced), thick walls, and two elongated, cylindrical cells, equal in length or one shorter, acute at the apex, with thick walls, completely united with each other on their longitudinal walls or diverging at the apex (Figs. 4e; 5d). Found exclusively in the cypselae, mainly on the ribs.
The nature of the indumentum in phyllaries and peduncles impart a different appearance to the surface, which can be observed at the light microscope and have taxonomic importance. Generally, phyllaries, peduncles, and smaller surrounding bracts share the same indumentum. The glandular hairs, short and straight, are somewhat perpendicular to the surface. The conical hairs, long and articulate, are incurvate, almost parallel to the surface. Sometimes the glandular hairs are mixed with the conical hairs.

Stevia cinerascens
Stevia cinerascens Baker is based on specimens collected in Brazil. Baker (1876) cited three original materials, two from southern Brazil (Sello 73 and Sello 74 and one from Porto Alegre (Fox s.n.). In the absence of the indication of a single specimen as the type, all the specimens cited in the protologue have to be treated as syntypes (Art. 9.5 of ICN, McNeill et al. 2012).
We found materials collected by Sello that fit accurately with the protologue deposited in K and P, and a photo kept at F of a material in B that is currently destroyed. In particular, they are one sheet labelled "Sello 74" in P (P 00704314), and three sheets "Sello 1949 (73)" that where found one in K (K 000488750) and two in P (i.e. P 00704312, Rodriguésia 70: e01532018. 2019 P 00704313). In addition, according to Stafleu & Corwan (1976-1988, the types of British Botanist J.G. Baker are kept mainly at K where he has worked. Thus, the specimen at K (K 000488750) is herein designated as the lectotype.
This species is distributed in Northeastern Argentina, Southern Brazil and Uruguay. It is recorded from Río Negro, Rivera, Soriano, Tacuarembó and Treinta y Tres departments (Fig.  8), uncommon, associated with hills and slopes, in calcareous and sandy soils.

Stevia congesta
This species is distributed in Southern Brazil (Rio Grande do Sul) and Southern Uruguay. It is only recorded from the Maldonado department (Fig. 8), growing on hills, in rocky soils.

Stevia gratioloides
It has not been possible to measure some florets traits. Other specimens assigned to this species by previous authors (e.g., Berro 2482, MVFA) have been herein identified as S. aristata.
This species is distributed in Southern Brazil and Northern Uruguay. It is only known from the original collection. It is important to mention that Banda Oriental was indicated on the label of the original material. Banda Oriental (or Banda Oriental del Uruguay) was the former name for the current Republic of Uruguay. However, the northern boundaries of Uruguay with Brazil at that time (approximately 1832) were imprecise and part of what was considered Banda Oriental, nowadays belongs to southwestern Rio Grande do Sul state. Therefore, Stevia gratioloides is included with doubts among the species of Uruguay until new collections corroborate its presence in the country.
On the other hand, S. entreriensis and Dissothrix hassleriana Chodat (1901: 411) constitute new synonyms of S. hirsuta, on the basis of similarities in the original descriptions and leaf blade shape, number of pappus bristles and pubescence. The name S. hirsuta has priority on S. entreriensis based on the ICN. This species is distributed in Centraleastern Argentina, Paraguay and southwestern and central Uruguay. It has been recorded as frequent in Colonia, Río Negro, San José, and Soriano departments (Fig. 8), and uncommonly in Durazno, Montevideo, Salto and Tacuarembó, inhabiting natural or anthropic shores and riverbanks, in dry or sandy soils.
Currently, Stevia megapotamica DC. (1836: 123) is included in the synonymy of Stevia multiaristata Spreng. (1826: 449). We found two sheets corresponding with the original material cited by Candolle, one deposited in P (P 00704368) and a fragment in G (G 00465268). Both sheets correspond with the information cited in the protologue, but since the material in P is more complete is herein designated as the lectotype.
This species is distributed in Central-eastern Argentina, southern Brazil in Rio Grande do Sul, and Uruguay. It is common throughout all Uruguay (Fig. 8) not recorded only in Río Negro department, growing on hills and hillsides, in rocky soils.
This species is distributed in Southern Paraguay and central Uruguay. It has been only recorded from Florida and Treinta y Tres departments (Fig. 8), rare, growing in riverbanks, associated to sandy soils.
Currently, this species shows three varieties (Freire & Ariza Espinar 2014): S. satureiifolia var. satureiifolia from central and northwestern Argentina, southwestern Brazil and Uruguay, and S. satureiifolia var. patagonica and S. satureiifolia var. ventanensis from central Argentina. According to our analysis, the uruguayan specimens belong to var. satureiifolia, since var. patagonica is characterized by its sessile heads (versus pedunculate heads in var. satureiifolia), and var. ventanensis by its leaf blades reaching 0.2-0.8 cm wide (versus 0.1-0.2 in var. satureiifolia).
During the analysis of S. satureiifolia, we detected that its synonym Stevia linariifolia DC. (1836: 123) needed lectotypification. It is based on original material gathered by Gaudichaud from Rio Grande. We found two specimens, one deposited in P (P 00704461) and other one in G (G 00465267), that fit accurately with the protologue. The specimen in P is herein designated as lectotype since it is a more complete plant.
This species is distributed in Central-eastern Argentina, southern Brazil, and Uruguay. It is very frequent in the southern Uruguayan departments (i.e., Colonia, Maldonado, Montevideo, Rocha, Soriano) (Fig. 8), and rare northwards, on hillsides, associated with dry environments, growing in rocky soils.
In the protologue of Kleinia selloi Spreng., Sprengel cited "Rio Grande. Sello" as the original material. We found four sheets kept at P that fits with the original description and some data of the protologue. In three of them (P 02677864, P 02677868, and P 02677873) the labels indicate that the plants were collected by Sello but in Brasilia (ancient name of Brazil). On the other hand, the fourth sheet (P 00704320) was collected in Rio Grande but did not have indication about the collector or its number. Since there is an absence of a designated type, all of these specimens have to be considered as original materials (Art. 40 Note 1 of ICN, McNeill et al. 2012;McNeill 2014). We designate here the specimen P 00704320 as the lectotype because fits accurately with the morphological description, it is a more complete plant and the type locality is indicated in its label.
Variety S. selloi var. ypacarayensis B.L. Rob (1930: 20) was based on material gathered by Hassler for two localities from Paraguay: (1) "on plains, Cordillera de Altos, Hassler, no. 3910" and (2) "in the region of lake Ypacaray, Hassler, no. 12,154". Original materials were found deposited in BM (BM 000096215, Hassler 3910), GH (GH 00012900, Hassler 12154), and US (US 00146085, Hassler 12154). We selected the specimen in GH as the lectotype, since fits accurately with the original description and it is a more complete plant.
Stevia veronicae (Fig. 7c) and S. oxylaena were described by Candolle (1836), in the same page, listed with the numbers 61 and 63, respectively. According to the original description, S. oxylaena (1836: 123) was morphologically similar to S. veronicae (1836: 123), but both species were distinct by several traits: internodes length (1-2.5 cm long in S. veronicae versus 0.5-2 cm in S. oxylaena), leaf blade shape (elliptical to obovate versus linear to elliptical, respectively), type of the secondary inflorescence (corymbiform cyme versus paniculiform cyme), and the number of pappus bristles (15-16 versus 20). From the first and second cited characters, there is overlap of measures. In addition, as a result of our analysis, intermediate variants among 15 to 20 pappus bristles were also found. In the case of the inflorescence, similar arrange of capitula were found in both species, with sessile or shortly pedunculate capitula (0.1-0.5 cm). Thus, we considered these species as synonyms.
We proposed S. oxylaena to be a synonym of S. veronicae because the latter is more frequently used in literature (e.g., Orth Ritter et al. 2010;Magalhaes et al. 2013;Soejima et al. 2017). Three specimens in total, kept at G and P that match the protologue of S. veronicae were found. Since Candolle's original materials are deposited in G (Stafleu & Corwan 1976), the sheet G 00465265 is designated herein as lectotype. As for S. oxylaena, two specimens were found kept at G (G 0046527) and P (P 00704319) that match the protologue. For this case, we selected the material kept at P since it fits accurately with the information of the protologue and it is a more complete plant.
In 1934, B.L. Robinson described S. ophryodonta (1934: 7) from northeastern Uruguay. The original material (GH 00012871) is scarce, consisting of fragments of five florets with a photograph of a complete specimen; to this date, we could not find where the photographed material is currently deposited. According to the original description, the leaf margin is dentate-serrate even though it appears entire at glance. The rest of traits that were utilized for delimiting this species, i.e. pubescence, phyllotaxis, leaf blade shape and margin, number of heads per inflorescence, and number pappus bristles, fit accurately with S. veronicae. Thus, we consider both species as synonyms.
This species is distributed in Central and southern Brazil and Uruguay. It has only been recorded in Maldonado and Rivera departments (Fig. 8), rare, growing on riverbanks, slopes and wetlands, in sandy soils.
Iconography. Fig. 9. More recently, this species was cited also from Uruguay on the basis of the specimen Chebataroff 5318, LP (Freire 2008). However, this specimen corresponds to S. selloi. As a result, S. burkartii is excluded here from Uruguay and becomes an endemic species of Argentina (Cabrera & Freire 1997;Freire & Ariza Espinar 2014) only known from its type material. Herter, Revista Sudamer. Bot. 6: 200. 1937. Nomen nudum. This name, cited by Herter (1937) without a description, is a nomen nudum. In his work Herter mentioned a specimen from the Tacuarembó department that is currently kept at Z (i.e. Z 000003929). This specimen fits with S. hirsuta by leaf and capitula characters.