A new report of phytomelanin in cypselae of Vernonieae: the case of the type species of Lychnophora Mart

Abstract

Phytomelanin is a mechanically hard, blackish, and inert substance rarely found in plants. In Asteraceae, this substance was historically associated with the Heliantheae alliance, but recent studies have observed it in unrelated groups as Heterocoma and Wunderlichia. During a taxonomic investigation, we found phytomelanin in cypselae of Lychnophora salicifolia an unusual feature in Vernonieae previously found only in Heterocoma. Furthermore, phytomelanin fills the intercellular spaces of the sclerenchymatic outer mesocarp in L. salicifolia. Our results doubt the (syn)apomorphy status in Heterocoma, suggest the phytomelanin may have not the same evolutionary significance in Lychnophorinae as in other tribes and proposes new perspectives for evolutionary studies in Asteraceae.

Key words
Compositae; calcium oxalate crystals; fruit anatomy; Lychnophorinae; micromorphology

INTRODUCTION

Cypselae are typical fruits found in Asteraceae (Marzinek et al. 2008), with high taxonomic value at different hierarchical levels (Marzinek et al. 2010, Marzinek & Oliveira 2010, Pandey et al. 2014, Tadesse & Crawford 2014, Franca et al. 2015, Silva et al. 2018, Bonifácio et al. 2019). One of the most notable features of the cypselae is phytomelanin, a brown to black non-cellular layer, hard, and resistant to degradation (Stuessy & Liu 1983), classically associated with the Heliantheae alliance pericarp (Misra 1964, 1972, Pullaiah 1979, 1981, Pandey & Singh 1982, 1983, 1994, Stuessy & Liu 1983, Pandey et al. 1989, Pandey 1998, Pandey & Dhakal 2001, Baldwin 2009). However, recent studies have reported unrelated groups (Freitas et al. 2015, Bonifácio et al. 2019), suggesting that phytomelanin is underestimated (De-Paula et al. 2013). From its earliest reports (Hanausek 1902, 1907, 1912) to the present day (i.e., De-Paula et al. 2013, Pandey et al. 2014, Tadesse & Crawford 2014, Franca et al. 2015, Freitas et al. 2015, Lusa et al. 2018, Silva et al. 2018, Bonifácio et al. 2019, Mathur & Pandey 2020), Synantherologists have been questioning the evolutionary significance of phytomelanin in Asteraceae fruits.

During taxonomic studies on Brazilian Vernonieae, we observed cypselae in Lychnophora type species were black due to the phytomelanin as in Heterocoma. As both genera are circumscribed in Lychnophorinae (Loeuille et al. 2015) and have cypselae with phytomelanin, we evaluated its deposition pattern in Lychnophora salicifolia Mart. and compared it with Heterocoma species, reassessing the systematics significance of the phytomelanin in Lychnophorinae and Asteraceae fruit evolution.

MATERIALS AND METHODS

We analyzed the cypselae of nine specimens of Lychnophora salicifolia (Table I). For the morphological study, cypselae were mounted on aluminum stubs and coated with gold using a sputter coater (Leica EM SCD050). The cypselae were observed using scanning electron microscopy (Zeiss EVO MA 100), and the images were digitally acquired.

Table I
List of Lychnophora salicifolia specimens examined.

Locality	Voucher information
Brazil, Brasília, Norte de Brazlândia	R.F. Vieira 2613 (HUFU)
Brazil, Brasília, Alto Paraíso de Goiás, Parque Nacional Chapada dos Veadeiros	R.F. Vieira 2560 (HUFU)
Brazil, Minas Gerais, Parque Nacional Chapada dos Veadeiros, trilha para Mulungu no parque	A.A.A. Barbosa et al. 169 (HUFU)
Brazil, Minas Gerais, Rio Pardo de Minas	A.C. Sevilha et al. 4342 (HUFU)
Brazil, Minas Gerais, Parque Estadual do Rio Preto	D.A. Chaves 375 (HUFU)
Brazil, Minas Gerais, Serra do Cipó	H.C. Sousa s.n. (HUFU 56703)
Brazil, Minas Gerais, Serra do Cipó	H.C. Sousa s.n. (HUFU 56704)
Brazil, Minas Gerais, Serra do Cipó	H.C. Sousa s.n. (HUFU 56706)
Brazil, Minas Gerais, Serra do Cipó	H.C. Sousa s.n. (HUFU 58732)

For the anatomical study, cypselae were rehydrated in a 5N NaOH solution for four hours (Anderson 1963, modified), dehydrated in an ethanol series, and embedded in historesin following the manufacturer’s protocol (Leica Microsystems). The cypselae were sectioned at 10 μm thickness using a rotary microtome (Leica RM 2235) and stained with 0.05% toluidine blue (O’Brien et al. 1964, modified). The sections were mounted in synthetic resin (Entellan), observed under a light microscope (Olympus BX51 with DP70 digital camera attached) and the images were also digitally acquired. The results were described following Roth (1977), wherein the exocarp originates from the ovarian inner epidermis, the endocarp from the inner epidermis, and the mesocarp from fundamental and vascular tissues. The endocarp and the innermost layers of the parenchymatic mesocarp are commonly crushed and consumed by seed growth.

RESULTS

Morphology

All specimens analyzed have cylindrical cypselae with well-developed ribs (Figure 1a) and twin hairs (zwillingshaare) among them. The carpopodium is conspicuous and symmetrical. The pappus is biseriate with a paleaceous and persistent outer series and a caducous inner series.

Figure 1
Cypselae morphology of Lychnophora salicifolia. arrow= phytomelanin; arrowhead= calcium oxalate crystal; tr= trichome; vb= vascular bundle. (a) General view (SEM), (b–c) transversal sections, (b) general view, (c) detail of the pericarp showing the phytomelanin and some crystals.

Anatomy

The pericarp of L. salicifolia possesses a uniseriate exocarp, and the mesocarp is divided into two regions, both with several layers. The outer mesocarp is composed of lignified cells (sclerenchyma) with phytomelanin filling its intercellular spaces. The inner mesocarp is parenchymatic and partially consumed by the seed development, which also absorbs the endocarp. Calcium oxalate crystals were observed in the outer mesocarp cells (Figure 1b–c).

DISCUSSION

The cypsela of L. salicifolia was similar to Heterocoma species, differing by indument, pappus, and mesocarp. Externally, the cypsela of L. salicifolia possesses twin hairs among the ribs, while Heterocoma is glabrous (Freitas et al. 2015). Regarding the pappus, the outer series of L. salicifolia is conspicuous and reduced in most species of Heterocoma, except H. albida (Freitas et al. 2015). Internally, the pericarp of Lychnophora salicifolia differs from Heterocoma by possessing calcium oxalate crystals in the mesocarp. The two most significant novelties of L. salicifolia were the occurrence of phytomelanin and the latter with calcium oxalate crystals simultaneously. Robinson (2009) noted that cypselae with phytomelanin never contain crystals, resulting from calcium oxalate crystals can be antagonistic to the phytomelanin production (King & Robinson 1987) or used in its production/secretion. This can be seen in the Heliantheae alliance; for example, the Helenieae is the only tribe that does not have phytomelanin and possesses crystals (Baldwin 2009). Nevertheless, this is the first report of the phytomelanin and crystals occurrence in the pericarp of Asteraceae concomitantly.

The phytomelanin was previously reported in stems of L. salicifolia by Lusa et al. (2018) when studied the evolutionary implication of phytomelanin in shoots of Lychnophorinae species. The reconstruction of ancestral character states indicated the most recent common ancestor of Lychnophorinae probably had phytomelanin in a thickened stem. However, the occurrence of phytomelanin in young stems and leaves would have appeared later, occurring independently in various taxa throughout the evolution of the group (Lusa et al. 2018). Also in Lychnophorinae, the phytomelanin in the stems could indicate that this pigment appeared first in the stem and was later incorporated into fruits (Lusa et al. 2018). This fact, along with the other phytomelanin distribution patterns among cypselae of Asteraceae studied until now, supports the hypothesis that fibers are responsible for the secretion of phytomelanin, as hypothesized by De-Paula et al. (2013).

The phytomelanin deposition pattern has presented a higher systematic value than only its occurrence. Freitas et al. (2015) reviewed the structure of fruits with phytomelanin and found three patterns distributing the Asteraceae pigment. The Eupatorieae pattern, in which there is a separation between the outer (parenchymatic) and inner (lignified) mesocarp, generates a schizogenous space filled by phytomelanin (Pandey & Singh 1983, 1994, Marzinek & Oliveira 2010, De-Paula et al. 2013, Freitas et al. 2015). In the Heliantheae pattern, no tissue separation and secretion occurs between the inner (parenchymatous) and internal (lignified) cells of the mesocarp (Misra 1964, 1972, Pandey & Singh 1982, 1994, Stuessy & Liu 1983, Pandey 1998, Julio & Oliveira 2009, Frangiote-Pallone & Souza 2014, Pandey et al. 2014, Freitas et al. 2015, Mathur & Pandey 2020). In the Heterocoma pattern, the phytomelanin occurs between the outer (lignified) and inner (parenchymatous) mesocarp (Freitas et al. 2015). Posteriorly, a different pattern was found by Bonifácio et al. (2019) in species of the genus Wunderlichia (Wunderlichieae, Asteraceae). In this pattern, phytomelanin is secreted between exocarp and mesocarp and around the phloem fibers. The phytomelanin secretion patterns are conservative in all the taxa studied until now (Freitas et al. 2015, Bonifacio et al. 2019). Both L. salicifolia and Heterocoma share a common pattern, and both are members of the subtribe Lychnophorinae. These patterns are relevant to studies with an evolutionary approach since they indicate homoplasy in the subtribe since, at the generic level, Heterocoma and Lychnophora are thought to lack a common ancestor (Loeuille et al. 2015).

CONCLUSIONS AND PERSPECTIVES

Despite the increasing number of phylogenetic studies, morphological and anatomical data are still essential for understanding the Asteraceae lineages. For many years, phytomelanin has been considered a (syn)apomorphy to the Heliantheae alliance, the latest divergent Asteraceae group (Panero & Funk 2008). However, recent anatomical studies (Fritz & Saukel 2011, Pandey et al. 2014, Freitas et al. 2015, Lusa et al. 2018, Bonifácio et al. 2019, Mathur & Pandey 2020, this study) have shown that the phytomelanin in this family is more widely distributed. Our results reinforce the idea that phytomelanin is homoplastic in Asteraceae and raises doubts about how conserved it may be the phytomelanin deposition pattern found in Lychnophorinae. Consequently, a more extensive study should be conducted to evaluate the importance of cypselae in the systematics and evolution of this subtribe.

ACKNOWLEDGMENTS

The authors thank the Laboratório Multiusuário de Microscopia Eletrônica of the Faculdade de Engenharia Química (UFU) for SEM facilities. The first author to thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Proc. 301734/2020-8) and Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico (Funcap Proc. DC8-0145-00117.01.00/18 and 9973919/2018) for the scholarship granted. The authors declare no conflicts of interest.

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Publication Dates

• Publication in this collection
  29 Sept 2021
• Date of issue
  2021

History

• Received
  12 Feb 2021
• Accepted
  4 July 2021