Comparative cypsela morphology in Campuloclinium DC. and contributions to Eupatorieae tribe (Asteraceae) systematics

: Cypselae anatomical studies have helped to understand the evolution and classification of some groups within Asteraceae. In Eupatorieae, there are many uncertainties about the Campuloclinium circumscription. There are currently two classifications for the genus, and still no consensus for their delimitation. Since structural studies have contributed to the delimitation of groups in Asteraceae, we studied the cypselae of Campuloclinium , searching how the pericarpial taxonomic features could enlighten the genus classification. We studied the fruits of eleven species of this genus through morphological and anatomical observation. Our results showed relevant features to the classification of Campuloclinium and its closely related groups. The stipitate cypsela together with other diagnostic characters are relevant to delimitation of this genus within of Eupatorieae. The trichomes present in cypselae have taxonomic proved to be a possible diagnostic character for the genus, and the six-celled trichomes are essential to distinguish C . campuloclinioides and C . hirsutum . The combination of phylogenetic and structural studies may lead to future research on the delimitation of Campuloclinium and its clades and understand how the stipitate cypselae and


INTRODUCTION
Campuloclinium DC. is a genus in the Eupatorieae tribe within Asteraceae (King & Robinson 1972, 1987).It presents a Neotropical distribution and comprises 12 species, which occur in southeastern South America, especially in Brazil (Calvo & Roque 2018).
The genus was described by Candolle (1836), and since then, it has undergone several modifications in its generic classification.It has been recognized as a section within Eupatorium L. (Bentham 1873, Baker 1876, Robinson 1918), and at others as a separate genus (Candolle 1836, Gardner 1846).However, Campuloclinium is currently recognized at the generic level (King As King & Robinson (1972, 1987) mentioned, reproductive structures have great importance to the classification of Campuloclinium.The fruits of Asteraceae, known as cypselae, arise from the inferior ovary (Marzinek et al. 2008).The cypselae have a high classification value in different hierarchical levels in the family (Angulo et al. 2015, Freitas et al. 2015, Via do Pico et al. 2016, Silva et al. 2018, Marques et al. 2018a, b, 2020, Bonifácio et al. 2019, Grossi et al. 2020) and the anatomical studies of these structures have contributed to solving taxonomy problems (Marzinek & Oliveira 2010, Freitas et al. 2015, Silva et al. 2018, Marques et al. 2018a, b, 2020, Bonifácio et al. 2019).
In Eupatorieae, anatomical studies with the cypselae have contributed to the resolution of taxonomic issues at the subtribal (Silva et al. 2018), generic (Marzinek & Oliveira 2010), and specific levels (Franca et al. 2015).Moreover, these studies have reevaluated the morphological terms of cypselae that sometimes generate confusion (Marzinek et al. 2010).
In this study, we evaluated the taxonomic value of cypselae for the classification of Campuloclinium.We compared the cypselae anatomy between species in the genus and then to other genera and subtribes within Eupatorieae to find features that would support some current classifications.

MATERIALS AND METHODS
The cypselae of 11 Campuloclinium species were studied using light microscopy and scanning electron microscopy (SEM).The material was obtained from herbaria of the Northeast Botany Institute (CTES, Argentina), the New York Botanical Garden Herbarium (NY), in NY, United States and the Federal Universities of Brasília (UB), Uberlândia (HUFU), and Bahia (ALCB), in Brazil.Details of the collection locations and the vouchers are available in Table I.For the SEM analysis, some samples were coated with gold and others were dehydrated in acetone and immersed in CO 2 to a critical point before gold coating.After that, all the cypselae were examined and photographed using the scanning electron microscopes JEOL 5800 LV or Zeiss EVO MA 100.
For observation of microcharacters, cypselae were softened in boiling water with a drop detergent, mounted in Hoyer's solution (Anderson 1954, King & Robinson 1970), and then analyzed under a Zeiss Axioplan light microscope.
For the anatomical analysis, fruits were rehydrated with a solution of 5M NaOH for half an hour (Anderson 1963, modified), dehydrated in an ethanol series, and embedded in historesin (Leica Microsystems, Heidelberg, Germany).The samples were sectioned in a rotating microtome using 10µm thickness.The cuts were 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 and images from three regions of the cypsela (basal, median, and apical) were acquired under the microscope Olympus BX51.
For the distribution of trichomes, three cypselae of each specimen were analyzed.The term rare was used when the trichomes were present in up to 10% of the surface of the cypsela, the frequent term was used between 11% and 50% and the term abundant when the trichomes were present above 50% of the surface of the cypsela.
Trichome terminology was based on Metcalfe & Chalk (1950, 1979).The description of the pericarp and the trichome distribution were carried out in an ontogenetic way, as proposed by Marzinek & Oliveira (2010).

External morphology
All cypselae studied are stipitate and prismatic, with evident ribs and trichome throughout the pericarp (Fig. 1a-k, Table II).Biseriate tector trichomes are restricted to the ribs and are composed of four cells (Table II).In the apical and basal portion of cypsela of C. campuloclinioides (Fig. 2b) and C. hirsutum is found biseriate tector trichome with six cells (Table II).Glandular trichomes were observed in the interrib region, with four to six basal cells and a biseriate head with eight cells (Fig. 2c-d, Table II).In Table II, it is also possible to observe the distribution pattern of the trichomes which have been categorized as rare, frequent and abundant in all species.
In the basal region is located the carpopodium, which presented annular shape in all species (Fig. 2e-h).The apical region has a uniseriate pappus with bristles connate on the base (Fig. 2i-l).

Anatomy
Almost all species presented five evident ribs (Fig. 3a).However, in some specimens of C. chlorolepis, (Fig. 3b) the cypsela has six ribs and in specimens C. parvulum, the cypsela can present eight ribs (Fig. 3c).
In the rib region, we observed a collateral vascular bundle involved by fibers (Fig. 3g-i).The distribution of phytomelanin is internal to the bundles (Fig. 3g-i).In all species, the endocarp is also consumed by seed development.
The carpopodium has a uniseriate exocarp and lignified, with cells slightly elongated in the periclinal sense (Fig. 3j); in C. purpuracens and C. hirsutum are observed glandular trichome.The mesocarp presents isodiametric to elongated cells, which can be parenchymatic or lignified (Fig. 3j).In the central region is observed vascular bundles (Fig. 3j).
The pappus presents one series of lignified bristles connate in the base in all species (Fig. 3k).
In the apical region of fruit, the exocarp presents cells with a primary wall, flattened in the periclinal sense (Fig. 3l).More internally lignified cells are found elongated in the periclinal sense (Fig. 3l).

DISCUSSION External morphology
As King & Robinson (1987) mentioned, all these cypsela features are important to delimitate this genus once these traits and the receptacle shape are diagnostic to Campuloclinium.Our study is in agreement with these authors and, again, the detailed study of cypselae corroborated the  importance of this reproductive organ for the classification of Asteraceae.
The presence of stipitate cypsela is important for generic delimitation in the two current possible phylogenetic relationships of this genus, one proposed by Robinson et al. (2009) and the second by Rivera et al. (2016a).In the first classification, Campuloclinium is closely related to Trichogonia (DC.) Gardner, with both genera sharing the presence of stipitate cypsela (Robinson et al. 2009, Roque et al. 2012) (2016b), in addition to the molecular data, the presence of a subplumose to plumose pappus and stipitate cypsela supports the classification of these genera into this new subtribe.In both analyses by Rivera et al. (2016a, b), a clade formed by Campuloclinium, Macropodina, and other genera in the Ayapaninae subtribe emerge as a sister group of a clade that contains Trichogoniinae and others subtribes.So, given these new phylogenetic analyses, is relevant to emphasize the importance of stipitate cypsela in the classification of Campuloclinium and possible related groups.Nevertheless, further research involving phylogenetic and ontogenetic studies is still necessary for understanding the evolution of the stipitate cypsela within these groups, once the delimitation of Campuloclinium within Eupatorieae is yet unclear.
In the cypsela, the biseriate tector trichome (twin hairs) are common in most Asteraceae genera (Hess 1938, Robinson 2009), but glandular trichomes can also be found (Marzinek & Oliveira 2010, Angulo et al. 2015, Via do Pico et al. 2016, Silva et al. 2018, Marques et al. 2018a, b, 2020, Grossi et al. 2020).The presence or absence of tector and glandular trichomes has successfully been used for the delimitation of genera and species in several groups within Asteraceae, including Eupatorieae (Marzinek & Oliveira 2010, Angulo et al. 2015, Freitas et al. 2015, Via do Pico et al. 2016, Silva et al. 2018, Marques et al. 2018a, b, 2020, Grossi et al. 2020).In Campuloclinium, all species present tector trichomes in ribs and glandular trichomes in interribs.Marzinek & Oliveira (2010) had already reported the trichome distribution pattern in Campuloclinium macrocephalum.This distribution pattern in Campuloclinium cypsela in conjunction with other characteristics can assist in genera identification at a specific level, type of trichomes and its distribution is very important to identification of species.The tector trichome with six cells is unique to C. campuloclinoides and C. hirsutum, and it can be used to differentiate both species within the genus.Distribution and trichome type are very important in Campuloclinium to species delimitation.The tector trichome with six cells is unique to C. burchelli, C. campuloclinoides and C. hirsutum, and this type of trichome could be a synapomorphy for these last two species taking into account the phylogeny performed by Rivera et al. (2016a).However, C. hirsutum is unique species with six cells tector trichome presents in the interrib region.Campuloclinium riedelii is a unique species that has cypsela with only glandular trichomes, while C. macrocephalum has only tector trichome, such characteristics differentiate both species from the others of the genus.Finally, C. hirsutum and C. purpuracens are the only species of the genus that present glandular trichome in the carpopodium.
The carpopodium is an abscission zone between the cypsela and the receptacle (Roque et al. 2009).According to Haque & Godward (1984), this region can delimitate genera in Asteraceae.The carpopodium symmetry is of great taxonomic value to some tribes (Freitas et al. 2015, Silva et al. 2018, Grossi et al. 2020).In Eupatorieae, studies performed by Silva et al. ( 2018) demonstrated that all species within of Disynaphiinae have a symmetric carpopodium, except Disynaphia praeficta (B.L.Rob.)R.M.King & H.Rob.In these studies, the assymetric carpopodium and other cypsela characteristic found in D. praeficta supported the exclusion this species from the subtribe, as well as had been demonstrated by molecular data (Silva et al. 2018).The carpopodium in Eupatorieae, in general, is conspicuous, but in some species this structure can be undistinguishable, as presented in recent studies performed by Silva et al. (2018) andGrossi et al. (2020).However, in Campuloclinium, the carpopodium is present and conspicuous in all species, therefore it is a structure with no taxonomic value for species delimitation.

Anatomy
Most of the analyzed species of Campuloclinium presented five ribs.This result agrees with those of Grossi et al. (2020), who reported that having five ribs is a feature present in most Eupatorieae.However, according to Marzinek et al. (2010), species with broader capitula had cypselae with a more uniform number of ribs while species of narrower capitula caused greater pressure between the cypselae, changing their shape.In our studies, we corroborate the hypothesis of Marzinek et al. (2010), because the cypselae of C. chlorolepis and C. parvulum as previously reported by Calvo & Roque (2018) they are narrower, and in our results these fruits have a variable number of ribs.
Until now, the carpopodium anatomy has shown little variation within Asteraceae (Marques et al. 2020).In some cases, as reported by Silva et al. (2018) and Marques et al. (2018aMarques et al. ( , 2020)), the carpopodium exocarp lignification can be useful for species separation, but in Campuloclinium this character does not present taxonomic value because it occurs in all species.

CONCLUSION
The studies presented here have shown that the morphology and anatomy of cypselae can bring new perspectives to the cypselae evolution in Eupatorieae, as well as to the subtribal and generic classifications of this tribe.The morphological studies presented here corroborate the importance of the stipitate form of the cypsela together with other diagnostic characters (such as, hemispherical or conical, scrobiculate and glabrous receptacle) to delimit Campuloclinium within Eupatorieae, that is, stipitate cypsela, such as this feature could have evolved independently within the Trichogoniinae and Campuloclinium + Macropodina.However, phylogenetic studies with a larger sampling of Campuloclinium species must be carried out to understand their relation to these other clades.A new possible diagnostic trait presented to Campuloclinium is the trichome distribution in the cypsela, which is characterized by tector trichomes in the ribs and glandular trichomes in the interribs.The external position of the phytomelanin layer regarding the vascular bundles is also important for understanding the subtribal classification in Eupatorieae.So far, all studied subtribes have the phytomelanin layer internal to the vascular bundles, except for Disynaphiinae.The position of this layer in Campuloclinium, which is a group close to Disynaphiinae.Finally, the finding of six-celled biseriate trichomes only in C. campuloclinoides and C. hirsutum reinforces the importance of the cypselae features at a specific level.

Table I .
Continuation.