Fruit of Lepidaploa ( Cass . ) Cass . ( Vernonieae , Asteraceae ) : anatomy and taxonomic implications

Lepidaploa is a taxonomically complex genus of Vernonieae, which is difficult to delimit morphologically due to vegetative and reproductive characters that overlap with Chrysolaena and Lessingianthus. Anatomical studies of cypselae are taxonomically useful for delimiting subtribes, genera and species of Asteraceae, and especially within Astereae and Eupatorieae. Given the importance of cypselae to the taxonomy of tribes of Asteraceae, we searched for morphological patterns among the species of Lepidaploa. Using light microscopy, we analyzed fruits of 21 species of Lepidaploa to evaluate the taxonomic position of the genus and species of questionable placement in the group. Our results showed that the morphologies of the cypselae of species of Lepidaploa are homogeneous and similar to other species of Vernonieae. However, pappus vascularization and the number of mesocarp layers could be useful for differentiating the sister groups Chrysolaena, Lepidaploa and Lessingianthus, which present similar macroand micro-morphological, palynological and chromosomal characters. Also, the presence of glandular trichomes and idioblasts in the cypsela, and lignified cells in the carpopodium exocarp, can be used to separate closely related species.


Introduction
Vernonieae is one of the most species-rich tribes of the Compositae, comprising about 1500 species distributed in Africa, Asia and the Americas (Keeley & Robinson 2009).Although Vernonieae exhibits a high degree of morphological variability, the tribe is generally known for its alternate leaves, discoid heads with violet flowers, acute style branches and biseriate pappus (Keeley & Robinson 2009).The cypselae of Vernonieae are generally cylindrical or turbinate, sometimes prismatic, and brown (Semir et al. 2011;Marques & Dematteis 2014;Angulo et al. 2015;Talukdar 2015;Redonda-Martínez et al. 2017;Pico et al. 2016).The pappus can be biseriate with an outer short paleaceous series and an inner long bristle series (Marques & Dematteis 2014;Angulo et al. 2015;Redonda-Martínez et al. 2017;Pico et al. 2016), biseriate with two paleaceous series (one short and one long) (Semir et al. 2011), or it can be absent (Redonda-Martínez et al. 2017).
Due to the taxonomic importance of cypselae for the delimitation of subtribes, genera and species of Vernonieae, numerous anatomical studies have been performed with taxa of this tribe (Misra 1972;Pandey & Singh 1980;Mukherjee & Sarkar 2001;Martins & Oliveira 2007;Galastri & Oliveira 2010;Jana & Mukherjee 2013;Freitas et al. 2015;Talukdar 2015;Redonda-Martínez et al. 2017).Most of the African and Asian species of Vernonieae that have been studied anatomically have revealed that the anatomy of the cypselae is very important to the taxonomy valuable of the tribe (Mukherjee & Sarkar 2001;Jana & Mukherjee 2013;Talukdar 2015).Some American species have also been studied, and their anatomical characters have also proven to be important to the taxonomy of the group (Martins & Oliveira 2007;Galastri & Oliveira 2010;Freitas et al. 2015;Redonda-Martínez et al. 2017).
Vernonieae is also known for the uncertain taxonomic delimitations of its contained taxa (Funk et al. 2005;Keeley et al. 2007).Part of the difficulty in defining generic limits for this tribe has been due to the fact that Vernonia s.l.(ca.1000 spp.) encompasses a large number of species with many different vegetative and reproductive features (Keeley & Jansen 1994;Keeley et al. 2007).Robinson (1999) compiled previous studies on the genus and delimited several other genera from Vernonia s.l.based on palynological, macro-and micromorphological, and cytological data.
Chrysolaena, Lepidaploa and Lessingianthus are three closely related genera that were thus segregated from Vernonia s. l. (Robinson 1999;Keeley & Robinson 2009).However, the delimitation of these three genera from one another is still complex due to the superposition of macro-and micromorphological characters, such as the type of inflorescence, shape of the heads, number of florets, absence or presence of a basal node, and indument of florets and cypselae, for example.Martins & Oliveira (2007) and Galastri & Oliveira (2010) have been the only studies to provide anatomical data on Chrysolaena and Lessingianthus, while such studies with Lepidaploa are practically nonexistent, except for anatomical studies o the cypsela of Lepidaploa tortuosa (Redonda-Martínez et al. 2017).
The genus Lepidaploa contains about 150 species that occur in the Neotropics (Pruski 2017), with Brazil and Bolivia being the countries with the highest species richness (Redonda-Martínez & Villaseñor 2011).Some species of Lepidaploa that inhabit southern South America (Argentina, Bolivia, Paraguay and southern Brazil) are of uncertain taxonomic position due to the overlap of vegetative and reproductive characters among species.
Given the limited anatomical knowledge about American Vernonieae, we studied the anatomy of the cypselae of some species of Lepidaploa that inhabit southern South America.Therefore, this study describes and compares the anatomy of the cypselae of species of Lepidaploa with the aim of identifying morphological patterns for the genus and its included species.

Materials and methods
A total of 21 species of Lepidaploa (Cass.)Cass.from southern South America were sampled.Details, including voucher information, are provided in Table 1.
For anatomical analysis, dried mature and immature cypselae were rehydrated with 5 N NaOH solution (Anderson 1963, modified from 36 to 4 hours), dehydrated in an ethanol series and embedded in historesin (Leica Microsystems, Heidelberg, Germany).The samples were sectioned using a rotary microtome at 10 μm thickness.The material was then stained with 0.05 % toluidine blue in acetate buffer, pH 4.7 (O'Brien et al. 1964 modified) and mounted with synthetic resin.The slides were analyzed under a light microscope (Olympus BX41) and digital images acquired.For images of the pericarp, we used sections of the median region of the cypselae.
Endocarp: The endocarp is consumed in the middle region in most of the cypselae, except for L. balansae (Fig. 1H).Stomata are located in middle region of the fruit of L. setososquamosa (Fig. 7A).
Floral disk: The floral disk is in the apical region of the fruit where the pappus is inserted.In the analyzed species, the floral disk is formed of uniseriate exocarp, with periclinally elongated and juxtaposed cells (Fig. 7B, C).The Acta Botanica Brasilica -32(4): 642-655 periphery of the inner region of the floral disk is composed of anticlinally elongated cells, while its center is formed of lignified cells with crystals (Fig. 7B, C).
Pappus: The pappus of all the analyzed species is biseriate (Fig. 7D, E).In the median transverse section, the outer series has a fusiform shape, while the inner series is composed of rounded bristles (Fig. 7D, E).Both series of the pappus are formed by lignified rounded cells with projections and vascularized bristles (Fig. 7D, E).
Carpopodium: In all the species studied, the carpopodium is formed of uniseriate exocarp with periclinally elongated and juxtaposed cells (Fig. 7F, G).The wall of the cells of the exocarp, in most of the species, is lignified (Fig. 7F) (Tab.2), but in L. amambaia, L. argyrotricha, L. buchtienii, L. myriocephala, L. salzmannii (Fig. 7G) (Tab.2), and L. setososquamosa, this wall is not lignified.Internally, the carpopodium possesses rounded primary wall cells in the periphery, while in its center there is a vascular bundle.
Seeds: All seeds are immature, except in L. psilostachya and L. tarijensis.The exotesta of L. psylostachya (Fig. 5B) possesses U-shaped thickening, while in L. tarijensis (Fig. 6G) lignification occurs only in the anticlinal wall of the exotesta.All of the species have only one vascular bundle (Figs. 1G;2G;3G), which passes through the region of the raphe to the region of the chalaza.

Discussion
All of the species analyzed of Lepidaploa exhibited the same structural pattern of the pericarp that has been reported for other genera of Vernonieae as in Bolanosa  2017).However, different sizes for the epidermal cells in the ribs and inter-ribs is reported for the first time.The presence of idioblasts and glandular trichomes in some species is important for taxonomic differentiation, whereas biseriate non-glandular trichomes, which were found in all species, have no taxonomic value within Vernonieae.According to Isawumi et al. (1966), idioblasts are generally present in species of Vernonieae and they may be characteristic structures of the tribe.Previous studies report the presence of idioblasts in Asian and African species of Vernonieae (Mukherjee & Sarkar 2001;Basak & Mukherjee 2003;Jana & Mukherjee 2012;Mukherjee & Nordenstam 2012), and American species of the tribe (Angulo et al. 2015;Redonda-Martínez et al. 2017;Pico et al. 2016).Despite their taxonomic importance at the tribal level, idioblasts must be used with care in the delimitation of Table 2. Morphological and anatomical features of the fruit of the studied species of Lepidaploa.?= unknown.
Acta Botanica Brasilica -32(4): 642-655.October-December 2018 species.The presence of a glandular trichome in four species is an important taxonomic character for the differentiation of some entities.For example, among the closely related species L. amambaia, L. remotiflora and L. setososquamosa, the last can be differentiated because it is the only one of this group that possesses glandular trichomes on the cypsela.Another species that also possesses this type of trichome is L. chamissonis, differentiating it from L. argyrotrichia, which does not possess glandular trichomes in the fruit.
The outer mesocarp exhibited two patterns: layers of cells non lignified or layers of cells lignified.This variation may be due to different stages of fruit development since we analyzed immature and mature cypselae of eight species (L.canescens, L. mapirensis, L. myriocephala, L. pseudomuricata, L. remotiflora, L. setososquamosa, L. sordidoppaposa and L. tarijensis).Ontogenic studies of the fruit of Chrysolaena obovata (Galastri & Oliveira 2010) reported four stages of maturation and demonstrated the structural differences of the outer mesocarp in these stages.Galastri & Oliveira (2010) observed that the outer and inner mesocarp differs in shape and size, and that there is gradual lignification.We also observed the pattern found in Chrysolaena obovata in the eight species of Lepidaploa mentioned above.Crystals were observed only in the outer mesocarp of all species.In transversal section, these crystals have a quadratic or rectangular shape.The presence of crystals is common within Vernonieae (Misra 1972;Pandey & Singh 1980;Mukherjee & Sarkar 2001;Martins & Oliveira 2007;Galastri & Oliveira 2010;Redonda-Martínez et al. 2017).According to King & Robinson (1987), the production of crystals in fruits is possibly antagonistic to the production of phytomelanin, a black substance found in the cypselae of species of Eupatorieae (Robinson et al. 2009;De-Paula et al. 2013), Heliantheae (Baldwin 2009), and Heterocoma (Freitas et al. 2015) (Vernonieae).Considering the antagonism hypothesis of King & Robinson (1987), we did not find phytomelanin in the cypselae of the species of Lepidaploa studied here.
The outer mesocarp of all Lepidaploa studied is continuous, and the number of layers present in this region does not exhibit great interspecific variability.Therefore, this region does not have taxonomic value among species of Lepidaploa, as demonstrated for the genera and species studied by Redonda-Martínez et al. (2017).In relation to the taxonomic importance of the outer mesocarp at the generic level, Chrysolaena (as seen in C. obovata and C. platensis) possesses the same number of cell layers (Martins & Oliveira 2007;Galastri & Oliveira 2010) as its sister genus, Lepidaploa.Meanwhile, Lessingianthus brevifolius (Martins & Oliveira 2007), a species of the sister genus of Chrysolaena and Lepidaploa, exhibited only one layer of cells in the outer mesocarp, and so may represent a taxonomic difference between Chrysolaena + Lepidaploa and Lessingianthus, if this same pattern is confirmed in the other species of Lessingianthus.Although this character is sufficient to differentiate Lessingianthus brevifolius from the other two genera, the number of cell layers must be used with care in the taxonomy of the group, since there are records in the literature of variation in the number of layers in different regions of the same fruit, as reported for Cyanthillium cinereum (Tiagi & Taimni 1960;Pandey & Singh 1980), V. fasciculata, V. missurica (Pandey & Singh 1980), and V. anthelmintica (Misra 1972;Pandey & Singh 1980;Talukdar 2015).
Variation in the number of ribs compared to the number of vascular bundles of the cypselae was found among the species analyzed.The number of vascular bundles does not necessarily correspond to the number of ribs since there are ribs in the fruits of Lepidaploa that are undefined.Differences between the number of ribs and the number of vascular bundles were also found for Vernonia bainesii, V. cistifolia (Mukherjee & Sarkar 2001), V. galamensis (Jana & Mukherjee 2013), Cyanthillium cinereum (Pandey & Singh 1980), Chrysolaena obovata, Lessingianthus brevifolius (Martins & Oliveira 2007), and Chrysolaena platensis (Galastri & Oliveira 2010).According to Marzinek et al. (2010), the formation of ribs is related to the position occupied by the flower in the capitula, which would generate the variability in the ribs of Lepidaploa.Our anatomical studies show that the number of ribs must be used carefully in the delimitation of genera and species, because several taxonomic studies have used the number of ribs to separate species, and sometimes genera, within the Vernonieae.However, in Lepidaploa the number of ribs is not taxonomically informative, given that some ribs are not conspicuous and cannot be seen with external morphological study.
The shape of the cells and the type of cell wall of the inner mesocarp found in the present study is similar that found in other Vernonieae genera, such as Bolanosa  (Misra 1972;Pandey & Singh 1980;Mukherjee & Sarkar 2001;Jana & Mukherjee 2013;Redonda-Martínez et al. 2017).It is worth pointing out that the pericarp of species of Vernonieae is non-multiplicative, which is confirmed by the consumed inner mesocarp of the cypselae of, mainly, L. psylostachya and L. tarijensis.According to Martins & Oliveira (2007), the small size of the pericarp of Chrysolaena obovata and Lessingianthus brevifolius favors dispersion by anemochory because the reduced number of layers of this region makes the fruit lighter.The consumed of the inner mesocarp during fruit maturation was also observed for the species studied by Tiagi & Taimni (1960) and Misra (1972), and in recent works with Chrysolaena obovata and Lessingianthus brevifolius (Martins & Oliveira 2007) and Chrysolaena platensis (Galastri & Oliveira 2010).
The floral disk is similar to the some species of Vernonia s.s.(Pandey & Singh 1980), and in Chrysolaena platensis (Galastri & Oliveira 2010).There were no significant differences among the floral disks of the different species analyzed in the present study, with the exception that some possessed lignified exocarp lignified in this region.However, this difference in lignin deposition in the exocarp wall may be related to the degree of maturity of the fruit, as suggested for the region of the outer mesocarp.Galastri & Oliveira (2010) observed crystals on floral disks in Chrysolaena platensis, but Marzinek & Oliveira (2010) and Silva et al. (2018) described only phytomelanin in this structure of species of Eupatorieae.
The basal region known as carpopodium is present in all the species analyzed here.The carpopodium anatomy was previously studied only for Chrysolaena obovata, Lessingianthus brevifolius (Martins & Oliveira 2007) and Chrysolaena platensis (Galastri & Oliveira 2010).The carpopodium of Lepidaploa is similar to the carpopodium found in Chrysolaena (Martins & Oliveira 2007) and Lessingianthus (Galastri & Oliveira 2010).In other words, this structure presents a uniseriate exocarp and its interior has parenchyma cells containing a group of sclereids in the center.Among the anatomical features studied, lignification in the exocarp of the carpopodium is the one that proved most useful for differentiating species.Some species exhibited an exocarp with a non-lignified wall, while the exocarp of other taxa possessed lignification of this region.We observed this difference among cypselae at the same stage of maturation, which indicates that it can be a useful character for differentiating closely related species.For example, L. remotiflora, a close relative of L. amambaia and L. setososquamosa, is the only one that possesses lignified cells of the exocarp of the carpopodium, while the others lack lignification.This difference is also important for separating L. argyrotricha (carpopodium with non-lignified exocarp) from L. chamissonis (carpopodium with lignified exocarp).
The seeds of Lepidaploa provide important taxonomic data, especially the characters of the testa.The exotesta of the seeds of L. psylostachya and L. tarijensis possess a secondary wall in the anticlinal and periclinal walls, and only in the anticlinal wall, respectively.The presence of a secondary wall of the exotesta was also reported in Vernonia anthelmintica, which exhibited U-shaped lignification (Talukdar 2015).
It is worth mentioning that Jana & Mukherjee (2013) did not observe the presence of a secondary wall in the exotesta of the same specimen of V. anthelmintica, which could be related to the degree of maturity of the cypsela.In Chrysolaena platensis (Galastri & Oliveira 2010), the region of the mesotesta and endotesta are consumed in the mature seed, however, lignin deposition was observed in the stages prior to the collapse of this structure.
The pappus is an important modification of the sepals that acts in the dispersion of the fruit (Roth 1977).We observed lignified cells with projections in the bristles of Lepidaploa; both structures were also found in species of Eupatorieae (Marzinek & Oliveira 2010) and in Chrysolaena obovata and Lessingianthus brevifolius (Martins & Oliveira 2007) of Vernonieae.Similarly, the presence of vascular bundles in the interior of each bristle of Lepidaploa was also found in other genera of Eupatorieae (King & Robinson 1987;Marzinek & Oliveira 2010) and Vernonieae (Misra 1972).However, previous studies show that vascular bundles are absent in the bristles of Chrysolaena obovata, Lessingianthus brevifolius (Martins & Oliveira 2007) and Chrysolaena platensis (Galastri & Oliveira 2010).Although vascular bundles are absent in species of Chrysolaena and Lessingianthus, more species of these genera need to be studied in order to establish the taxonomic validity of this absence.According to Mukherjee & Sarkar (2001), the pappus is an important structure for the identification of genera and species.The species of Lepidaploa exhibited the same pattern for the pappus, and so the anatomy of this structure cannot be used as a taxonomic tool to separate these entities.On the other hand, the anatomy of the pappus could be important for distinguishing Lepidaploa from Chrysolaena and Lessingianthus.In Chrysolaena (Martins & Oliveira 2007;Galastri & Oliveira 2010) and Lessingianthus (Martins & Oliveira 2007), the bristles of the pappus do not possess vascular bundles, which would differentiate these two genera from the closely related genus Lepidaploa, which possesses vascular bundles in the pappus.

Conclusion
The cypselae of Lepidaploa species exhibited biseriate trichomes, uniseriate exocarp, outer mesocarp (generally with one or two layers of cells and the presence of crystals), and vascular bundles in the inner mesocarp; this morphology is similar to that of other taxa of Vernonieae.However, there are important taxonomic differences between species in relation to idioblasts, glandular trichomes, lignification of the carpopodium, and pappus vascularization.The significance of carpopodium lignification for identifying species of Lepidaploa is reported here for the first time.In addition, the pappus vascularization and number of mesocarp layers could provide important taxonomic data for segregating Chrysolaena, Lepidaploa and Lessingianthus genera.

Figure 4 .
Figure 4. Transversal sections of the fruit of the studied species of Lepidaploa: A-B.L. myriocephala; C-D.L. novarae, in D detail of idioblasts in the exocarp and detail of the crystals in the outer mesocarp; E-F.Immature cypsela of L. pseudomuricata; G-H.Mature cypsela of L. pseudomuricata.cr: crystals; id: idioblasts; se: seed; vb: vascular bundles.

Figure 7 .
Figure 7. Middle region of cypsela, floral disk, pappus, carpopodium and ribs of the studied species in transversal section.A. L. setososquamosa, detail of the stomata in the exocarp and endocarp; B. L. eriolepis, floral disk; C. L. salzmannii, detail of lignification (asterisk) in the floral disk; D-F.L. amambaia, outer and inner series of the pappus; F: L. beckii, detail of the carpopodium with lignified cells in the exocarp; G. L. salzmannii, detail of the carpopodium without lignified cells in the exocarp; H-I.L. balansae, detail comparing non-lignified outer mesocarp and lignified outer mesocarp.oi: inner series of the pappus; os: outer series of the pappus; pa: pappus; st: stomata; tr: trichome; vb: vascular bundles.