FRUIT FEATURES IN SOME SPECIES OF ASTEREAE AND HELINATHEAE ALLIANCE

Theoretical framework : Astereae is the second largest tribe of Asteraceae, with 170 genera with a worldwide distribution. The Alliance Heliantheae clade consists of about 160 genera from various tribes, including Eupatorieae and Heliantheae. Both taxa, especially Alliance Heliantheae, have taxonomic problems, and the structure of the fruits can reveal useful characters in their taxonomy. Objective : The fruits of Baccharis uncinella DC., Inulopsis scaposa (DC.) O. Hoffm. (Tribe Astereae), Campovassouria cruciata (Vell.) R. M. King & H. Rob. (Tribe Eupatorieae) and Ichthyothere mollis Baker (Tribe Heliantheae) were ontogenically analyzed and compared with results obtained in studies of species from these three tribes. Method : Flowers and fruits at different stages of development were collected in Vila Velha State Park, Paraná, Brazil. The botanical material was investigated according to usual techniques in plant anatomy. Results : The fruits of the two species of Astereae are structurally very similar, being devoid of mesocarp and phytomelanin, unlike the species of Eupatorieae and Heliantheae, which have parenchymatic and sclerenchymatic mesocarp and phytomelanin. Conclusion : Characters that may be relevant in the taxonomy of both groups of Asteraceae are the absence or presence of mesocarp; presence or absence of phytomelanin


INTRODUCTION
Astereae is a tribe of worldwide distribution of about 3000 species, and is considered the second largest tribe of Asteraceae (Funk et al., 2009).Heliantheae Alliance is made up of several tribes, but studies have shown that there is a need to rearrange the groups; Eupatorieae and Heliantheae tribes are nested in this alliance (Funk et al., 2009).
The morphology of the pappus and the structure of the cypselae can reveal characters that are important in the taxonomy of Asteraceae tribes (Judd et al., 2009).Marques et al. (2021) also recognized the high taxonomic value of cypselae in different groups of Asteraceae.Structural characters of pappus, cypselae and seeds have demonstrated significant taxonomic potential in the separation of species and genera, and in the characterization of different hierarchical groups of Asteraceae (Pandey & Singh, 1984;King, 1989;Mukherjee & Sarkarj, 2001;Julio & Oliveira, 2009;Marzinek & Oliveira, 2010;Pallone & Souza, 2014;Franca et al., 2015;Batista & Souza, 2017a,b;Akhlaq et al., 2022).
In the present work, four species were selected to study the pappus and the developing fruit, two from the Astereae tribe and two from the Heliantheae Alliance.The data obtained were compared with other species already studied belonging to the same groups (Table 1).Our objective was to show whether these characters can be diagnostic in the separation of species and tribes, and can also be useful in the phylogeny of both groups.

MATERIAL AND METHODS
Plant samples -Branches with floral buds, flowers and fruits at different stages of development of Baccharis uncinella DC., Inulopsis scaposa (DC.)O. Hoffm.(Tribe Astereae), Campovassouria cruciata (Vell.)R. M. King & H. Rob. (Tribe Eupatorieae) and Ichthyothere mollis Baker (Tribe Heliantheae) were collected in Vila Velha State Park, located in the municipality of Ponta Grossa, Brazil.Vouchers were deposited in the Herbarium of the State University of Maringá, Brazil, under registration numbers 29611, 24551, 24608 and 24588, and 24597, respectively.Analysis in light microscope -Buds, flowers and fruits were fixed in FAA 50 (Formaldehyde, Acetic Acid and 50% Ethyl Alcohol), embedded in Leica® historresin as recommended by the manufacturer, sectioned transversally and longitudinally in a rotation microtome and stained in toluidine blue (O'Brien et al., 1964).Photomicrographs were taken using a Leica EZ4D light microscope and subsequently processed using Leica Application Suite version 1.8 software.
Analysis in scanning electron microscope (SEM) -The micromorphological analysis (Bozzola & Russel, 1999) of the pappus and surface of the fruit of the four species was carried out on material fixed in FAA 50, dehydrated in an ethyl series and dried at the critical point of CO2 (Horridge & Tamm, 1969).Then, the samples were mounted on aluminum supports and covered with a layer of 30 to 40 nm gold.The analysis was carried out using a scanning electron microscope (SEM) model Shimadzu SS 550, with the scales of the electron micrographs directly printed on them.

RESULTS AND DISCUSSION
Our results on the analyzes of the ovaries and fruits (pappus and pericarp) of the four species investigated here are shown in Tables 2 and 3.The ovaries of the two species of Astereae have significant differences, with the glabrous external epidermis and homogeneous mesophyll in Baccharis uncinella, and with twin hairs in formation and mesophyll with two differentiable histological regions in Inulopsis scaposa (Figure 1B).Both species of Eupatorieae and Heliantheae, Campovassouria cruciata and Ichthyothere mollis, exhibit more similarities between them, in which the mesophyll of both species has three tissue regions (Figure 1C,D).Twin hairs were found only in Ichthyothere mollis (Figure 1D).The literature on Astereae reveals analysis of ovary tissues from only the species Conyza bonariensis, which consists of an outer epidermis with twin hairs and a parenchymatic mesophyll with two regions, one with radially elongated cells and the other with three to four cell layers.The cypselae of other Astereae species have been structurally investigated, but Mukherjee & Sarkarj (2001) did not analyze the anatomy of the ovary wall (Table 1).
The Eupatorieae species Campovassouria cruciata (Table 2) revealed ovary mesophyll with three different tissue regions (Fig. 1C), but mesophyll with two tissue regions has also been reported by Marzinek &Oliveira (2010), andBatista &Souza (2017a,b) in other species of the tribe (Table 1).It has been observed the outer mesophyll with two or more cell layers, and inner mesophyll that varies from three to six cell layers (Table 1) (Marzinek & Oliveira, 2010;Batista & Souza, 2017a,b).
The number of three tissue regions found in the ovary mesophyll of Ichthyothere mollis is ordinarily the same as the number found in other species of Heliantheae (Table 1).Both species of Bidens (Julio & Oliveira, 2009), Tridax procumbens (Pallone & Souza (2014), and Eclipta alba and Parthenium hysterophorus (Batista & Souza, 2017a,b) exhibit mesophyll with three types of parenchyma (Table 1).The cypselae of the four species investigated here have pappus (Table 3), being bristly in Baccharis uncinella, Inulopsis scaposa and Campovassouria cruciata, and laminar in Ichthyothere mollis (Fig. 2A-D).In the first three species, epidermal pappus cells have a tapered free end (Fig. 2A,C,E).The pappus (Fig. 2A,B) of the two Astereae species consists of epidermis and reduced mesophyll, mainly in Baccharis uncinella.Pappus vascularization is reduced in Inulopsis scaposa (Fig. 2D) and absent in Baccharis uncinella (Fig. 2B).Campovassouria cruciata (Eupatorieae) has a pappus similar to the two species of Astereae (Fig. 2F), but Ichthyothere mollis (Heliantheae) has a notably different pappus, with a laminar shape (Fig. 2G,H).The rudimentary vasculature of the pappus of these species and Astereae, associated with the reduction and sclerification of the mesophyll, reinforces the thesis that the pappus can be considered as a less differentiated form of the leaf, according to Roth (1977).Reduced vascular tissue in the pappus has been recorded in Conyza bonariensis, and in several species of Eupatorieae (Marzinek & Oliveira, 2010) and Heliantheae (Pallone & Souza, 2014) (Table 1).The cypselae of the investigated species have an epidermal, cuticularized exocarp with twin hairs (biseriate trichomes) in Baccharis uncinella, Inulopsis scaposa and Ichthyothere mollis.Campovassouria cruciata has a glabrous exocarp.These twin hairs are typical of many species of Asteraceae (Compositae) (Roth, 1977).Contrary to Roth's (1977) interpretation, Marzinek & Oliveira (2010) considered the biseriate trichomes to have taxonomic value, at least in Eupatorieae.These authors established four types of biseriate trichomes (two nonglandular and two glandular), which presented a variable number and shape of cells.Presence or absence of trichomes on the cypselar surface was considered of taxonomic value for seven Helianthus L. (Heliantheae) species (Basak & Mukherjee, 2001).Baccharis uncinella and Inulopsis scapose (Astereae) have the mature fruit devoid of mesocarp and endocarp (Fig. 3A,B), which are destroyed or collapsed during their development.In addition to the exocarp, vascular bundles located on the ribs or lobes of the pericarp remain in the mature fruit (Fig. 3A,B).The absence of mesocarp and endocarp appears to be common in many species of Astereae, as seen in Aster amellus, Aster peduncularis, Aster thomsonii, Baccharis halimifolia, Erigeron acer, Erigon villarsii, Myriaclis humilis and Vittadinia gracilis (Mukherjee & Sarkarj, 2001) (Table 1).
Considering the absence of a large part of the pericarp and phytomelanin in the ripe fruit in Astereae (Fig. 3A,B), it is likely that the protection of the embryo in species of this tribe, at least in Baccharis uncinella and Inulopsis scapose, is provided by the seed coat.It consists of uniseriate exotesta of thick-walled cells and other inner layers that are strongly compressed.Seed coat with specialized exotesta has already been registered in several other Asteraceae species (Pandey & Singh, 1984;Pallone & Souza, 2014;Batista et al., 2015).
Phytomelanin (Fig. 3C,D) recorded in the fruits of Eupatorieae and Heliantheae species is considered by Roth (1977) as a black or dark brown pigment that typically occurs in many cypselae, and appears to have the protective function of the pericarp and seed against excessive insolation.The presence of phytomelanin is classically associated with the Helliantheae Alliance, but recent research has reported phytomelanin in fruits of other Asteraceae taxa (Marques et al., 2021).The presence of phytomelanin in Asteraceae has long been considered a synapomorphy in the family (Panero, 2007).However, Marques et al. (2021) have shown that recent anatomical studies revealed that the phytomelanin in this family is more widely distributed.
The Astereae species analyzed here exhibit similar structural characters of the ovary and fruit, with insignificant taxonomic differences.On the other hand, Eupatorieae and Heliantheae species show differences in ovary contour and epidermis structure (Table 3).The differences in these last two tribes are still significant in relation to the fruit, mainly regarding the pappus, exocarp and mesocarp (Table 3).With reference to the three tribes, our studies (Table 3) and others carried out by different authors (Table 1) show that Astereae has significant differences when compared with Eupatorieae and Heliantheae, that is, in the former there is no mesocarp and phytomelanin in the ripe fruit, whereas in the latter the mesocarp with different histological regions and phytomelanin are present.Characters recorded in this study (Tables 2 and 3) and in similar investigations (Table 1), which may be useful in the taxonomy of the tribes are: absence or presence of mesocarp; presence or absence of phytomelanin, laminar or bristle pappus and occurrence or type of twin hairs.

CONCLUSION
Characters analyzed from Astereae fruits are not very significant for separating the species, showing structural uniformity in the tribe.On the other hand, the absence of mesocarp and phytomelanin is a relevant character to separate Astereae from the tribes belonging to the Heliantheae Alliance.The species investigated in this Alliance, which are part of the tribes Eupatorieae and Heliantheae, have characters related to the ovary, pappus, exocarp and mesocarp that are important for the diagnosis of the species.

Table 1 -
Structural characters of the ovaries and cypselae of species belonging to the tribes Astereae, Eupatorieae and Heliantheae, according to different authors.