Universidade Federal de Santa Maria

Ci. e Nat., Santa Maria, v. 43, e75, 2021

DOI: 10.5902/2179460X43417

ISSN 2179460X

Submitted: 04/08/2021 • Approved: 21/07/2021 • Published: 06/10/2021

Biology-Botany

Contribution to the identification of the species Myrcia hatschbachii D. Legrand (Myrtaceae): anatomical and histochemical analyses

Contribuição para a identificação da espécie Myrcia hatschbachii D. Legrand (Myrtaceae): análises anatômicas e histoquímicas

Larissa Junqueira GattoI

Vanessa Barbosa BobekII

Jane Manfron BudelIII

Natasha Tiemi FabriIV

Josiane de Fatima Gaspari DiasV

Obdulio Gomes MiguelVI

Marilis Dallarmi MiguelVII

I PhD in Pharmaceutical Sciences, Federal University of Parana, Curitiba, PR, Brazil

http://orcid.org/0000-0001-6451-7445 – lari.gatto@gmail.com

II PhD in Pharmaceutical Sciences, Federal University of Parana, Curitiba, PR, Brazil

http://orcid.org/0000-0002-2836-2076 – vanessabbobek@gmail.com

III PhD in Pharmaceutical Sciences, Professor of the Department of Sciences Pharmaceuticals, State University of Ponta Grossa, Ponta Grossa, PR, Brazil

http://orcid.org/0000-0003-1873-2253 – janemanfron@hotmail.com

IV Master in Pharmaceutical Sciences, Federal University of Parana, Curitiba, PR, Brazil

http://orcid.org/0000-0003-4237-7626 - natasha.fabri@gmail.com

V PhD in Pharmaceutical Sciences, Professor of the Department of Pharmacy, Federal University of Parana, Curitiba, PR, Brazil

http://orcid.org/0000-0002-8548-8505 – josianefgdias@gmail.com

VI PhD in Chemistry, Professor at the Department of Pharmacy, Federal University of Parana, Curitiba, PR, Brazil

http://orcid.org/0000-0002-2231-9130 – obdulio@ufpr.br

VII PhD in Agronomy, Professor at the Department of Pharmacy, Federal University of Parana, Curitiba, PR, Brazil

http://orcid.org/0000-0002-1126-921 – marilisdmiguel@gmail.com

ABSTRACT

The genus Myrcia is used in folk medicine to treat diabetes. The plants used in folk medicine require morphological and anatomical references to attest to its authenticity. This is the first report of the microscopic study of Myrcia hatschbachii. In this sense, the aim of the present study was to describe the anatomical characteristics, in order to contribute to the species’ identification. For anatomical and surface analyses free-hand sections were prepared and observed using optical microscopy, simultaneously some materials were processed and observed under scanning electron microscopy. In addition, histochemical tests were performed. The anatomical features described here correspond with previously reported features found in other species of the genus Myrcia. Furthermore, the following anatomical markers were observed in Myrcia hatschbachii: druses in the spongy parenchyma, concave-convex midrib, heart-shaped petiole, oval-shaped stem, C-shaped vascular bundle in the midrib and open arc shape with invaginated ends in the petiole; phenolic compounds in the phloem and lignified cells in the pith. The results of anatomical and histochemical analyses provide micromorphological and microchemical features that can help in the taxonomy and proper identification of the species.

Keywords: Microscopy; Microchemistry; Morpho-anatomy; Myrtaceae; Scanning electron microscopy

RESUMO

O gênero Myrcia é usado na medicina popular para tratar a diabetes. As plantas utilizadas na medicina popular requerem referências morfológicas e anatômicas para atestar sua autenticidade. Este é o primeiro relato do estudo microscópico de Myrcia hatschbachii. Nesse sentido, o objetivo do presente estudo foi descrever as características anatômicas, a fim de contribuir para a identificação da espécie. Para as análises anatômicas e de superfície, cortes à mão livre foram preparados e observados em microscopia óptica, simultaneamente alguns materiais foram processados e observados em microscopia eletrônica de varredura. Além disso, foram realizados testes histoquímicos. As características anatômicas descritas aqui correspondem às características relatadas anteriormente encontradas em outras espécies do gênero Myrcia. Além disso, os seguintes marcadores anatômicos foram observados em Myrcia hatschbachii: drusas no parênquima esponjoso, nervura central côncava-convexa, pecíolo em forma de coração, caule com formato ovalado, feixe vascular em C na nervura central e forma de arco aberto com extremidades invaginadas no pecíolo; compostos fenólicos no floema e células lignificadas na medula. Os resultados das análises anatômicas e histoquímicas fornecem características micromorfológicas e microquímicas que podem auxiliar na taxonomia e identificação adequada das espécies.

Palavras-chave: Microscopia; Microquímica; Morfoanatomia; Myrtaceae; Microscopia eletrônica de varredura

1 INTRODUction

Myrcia DC. belongs to the subtribe Myrciinae O.Berg (sensu LUCAS et al., 2019) and to the tribe Myrteae (sensu WILSON et al., 2005). It is one of the largest genera of Myrtaceae, comprehending 780 Neotropical species (SANTOS et al., 2016). In Brazil 411 native species of Myrcia can be found, especially in Brazilian cerrado and Atlantic Forest (LUCAS et al., 2011; Flora do Brasil, 2020). Several Myrcia species are called insulin plants, due to its folk use in the treatment of diabetes. This group of plants includes Myrcia punicifolia (Kunth) DC., Myrcia speciosa (Amsh.) Mc Vaugh, Myrcia amazonica DC., Myrcia citrifolia (Aubl.) Urb., Myrcia guianensis (Aubl.) DC., Myrcia multiflora (Lam.) DC., Myrcia salicifolia DC., Myrcia sylvatica (G. Mey) DC., Myrcia uniflora DC. (Cascaes et al., 2015). It is important to highlight that M. multiflora, known as “pedra-ume-caa”, is found in the market sold in the form of tea or capsules to treat diabetes (Brito; Lanetti, 1996). Additionally, species of Myrcia are traditionally used as astringents and diuretics and in the treatment of ulcers and hypertension (Alves et al., 2016).

The plants used in folk medicine require morphological and anatomical references to attest for its authenticity, since the morphological similarity or use of such popular names can lead to improper use of certain plant drug. In this sense, the American species of Myrtaceae are similar in most of the morphological characters, making their identification and classification difficult. Thus, the anatomical aspects assist the correct diagnosis of plant species, even when they are outside the flowering or fruiting period (Donato; Morretes, 2011).

Myrcia hatschbachii D. Legrand is a native and endemic tree from the Southern states of Brazil and presents Atlantic Forest domain (BFG, 2018). A study with the species showed a high content of phenolic compounds and the characterization of ellagic acid and 3-o-methyl ellagic acid. In addition, the extract exhibited antioxidant properties, by the methods of DPPH, FRAP, and ABTS, and relevant antifungal activity against Candida albicans (GATTO et al., 2020). Considering the potential therapeutic use of the species (GATTO et al., 2020), the morphological similarities in the family (Donato; Morretes, 2011) and the absence of anatomical studies published in the literature, the aim of the present study was to carry out an anatomical and histochemical analyses in order to contribute to the identification of M. hatschbachii and assist further studies on the possible differentiation of this species from other species of the genus Myrcia, including the insulin plants group since the morphological characterization between them is similar.

2 Materials and methods

2.1 Plant materials

Aerial vegetative parts of M. hatschbachii were collected in autumn, at the Cifloma Capon, located at the Federal University of Parana; Curitiba, Brazil (coordinates 25°26’S, 49°14’W). Species identification was done by a taxonomist, in comparison to specimen number 72379 held at the herbarium in the MBM. Access to the botanical material was approved and licensed by the Ministério do Meio Ambiente and registered under number 02001.001165/2013-47.

2.2 Light microscopy

Fresh leaves and stems (secondary structure) were fixed in an FAA solution (Johansen, 1940) and stored in 70% ethanol (Berlyn; Miksche, 1976). Semipermanent microscope slides were obtained through free-hand paradermal and cross sections (OLIVEIRA; AKISUE, 1997), then the sections were stained with Astra blue and basic fuchsine (O’BRIEN et al., 1964). The sections were then mounted on glass slides in a drop of 50% glycerin (BERLYN; MIKSCHE, 1976).

For the preparation of permanent slides, some of the FAA-fixed specimens were washed in distilled water and then dehydrated using a series of ethanol solutions (30, 50, 70, 95, and 100%) before passing through a graded series of xylene: ethanol solutions and then to 100% xylene. The specimens were then embedded in paraffin and transversely sectioned (7–9 μm thick) with a rotary microtome (Spencer 820). Paraffin from the sections was removed using xylene. The sections were then rehydrated, stained with astra blue/basic fuchsin (ROESER, 1972), and mounted in synthetic resin (Entellan®).

For the analysis of the leaf epidermal features, the leaf specimens were cleared by submersion in a sodium hypochlorite solution for 24 h. After the clarification, the material was rinsed with distilled water, a 5% acetic acid solution, and then rinsed with distilled water before being stained with 1% safranine (Fuchs, 1963) and then the slides were mounted on glass slides in a drop of 50% glycerin.

2.3 Histochemical tests

For the histochemical tests, freehand cross sections were made, after which the following reagents were used: potassium dichromate (Gabe, 1968) and ferric chloride (JOHANSEN, 1940) solutions for the identification of phenolic compounds; Sudan III for lipids (Sass, 1951); phloroglucinol/HCl to distinguish lignified elements (Foster, 1949); 1% iodine solution for starch (BERLYN; MIKSCHE, 1976); and sulfuric acid to determine the chemical composition of crystals (OLIVEIRA; AKISUE, 1997). The photomicrographs were taken using a light microscope (Olympus CX 31) equipped with a C7070 digital camera.

2.4 Scanning electron microscopy

Newly collected samples of leaves and stems were dehydrated in a series of ethanol solutions of increasing concentrations and then dried in a Leica EM CPD300 critical point dryer (Leica Microsystems, Wetzlar, Germany). Afterwards, the dried samples were mounted on aluminum stubs using glued carbon ribbon and then coated with gold using a Quorum SC7620 sputter coater (Quorum technologies Ltd., Kent, UK). Electron micrographs were performed using a Tescan Mira3 field emission SEM in high vacuum mode with an accelerating voltage of 15 kV. This procedure was carried out at the multi-user laboratory (C-Labmu) in the State University of Ponta Grossa (UEPG).

3 Results

In frontal view, the leaves of M. hatschbachii exhibit wavy anticlinal epidermal cell walls (Fig. 1b, c) and striated cuticle on both sides (Fig. 1d, e). The leaves are hypostomatic, and the stomata are paracytic (Fig. 1c). In cross-section, the epidermis is one-layered with cells more elongated in the anticlinal direction (Figs. 1f, 2a). The cuticle is thin on both surfaces and it reacts positively to Sudan III. Unicellular non-glandular trichomes can be observed (Fig. 2d).

Figure 1 – Morpho-anatomy of leaves of Myrcia hatschbachii

Source: Authors (2018)

In where: [b, c, and f: light microscopy; d-e: SEM]. (a) A shoot with leaves. (b–e) Leaf epidermis in surface view (b: adaxial side, showing the wavy anticlinal epidermal cell walls; c: abaxial side, hypostomatic leaves; d: adaxial side, evidencing the striated cuticle; e: abaxial side, evidencing the striated cuticle). (f) Transverse sections of the leaf blade. [ct, cuticle; ep, epidermis; pc, phenolic compounds; pp, palisade parenchyma; sp, spongy parenchyma; st, stomata; vb, vascular bundle]. Scale bar: a = 2 cm; f = 50 µm; d, e = 20 µm; b-c = 25 µm.

The mesophyll of leaves is dorsiventral, formed by a layer of palisade parenchyma (Figs. 1f, 2a) and approximately eight layers of spongy parenchyma. The cells of the palisade parenchyma have a high content of phenolic compounds, which reacted positively with the ferric chloride solution in the histochemical test (Fig. 2a, f). Minor collateral vascular bundles were located in the spongy parenchyma and were surrounded by a parenchymatic sheath (Fig. 1f). Several calcium oxalate druses were also found in the spongy parenchyma (Fig. 2a, b). The midrib has a concave-convex shape, with a prominent concavity on the adaxial side (Fig. 2c, f).

In the midrib region, the epidermis of leaves exhibits similar characteristics previously described for the lamina, except for being more densely covered by non-glandular trichomes. Beneath the epidermis, there are some layers of angular collenchyma on both sides. The vascular system is bicollateral (Fig. 2c, f, g, h), with a single C-shaped vascular bundle with the opening facing the adaxial side. The vascular system is surrounded by a continuous sclerenchymatic sheath (Fig. 2c, e), which reacts positively with phloroglucinol/HCl in the histochemical test. Several phenolic compounds are present in the phloem (Fig. 2e, h), and they react with ferric chloride (Fig. 2f) and potassium dichromate (Fig. 2g). The ground parenchyma contains secretory cavities (Fig. 2f), starch grains, and calcium oxalate druses (Fig. 2b).

Figure 2 – Anatomy of leaves of Myrcia hatschbachii

Source: Authors (2018)

In where: [a, c-h: light microscopy; b: SEM]. Transverse sections of the leaf blade (a, b) and the midrib (c-h). Phenolic compounds in reaction with ferric chloride (a, f) and potassium dichromate (g); Cuticle in reaction with Sudan III (d); Xylem in reaction with phloroglucinol/HCl (f). [ca, cambia; ct, cuticle; dr, druse; ep, epidermis; fi, fiber; gp, ground parenchyma; nt, non-glandular trichome; pc, phenolic compounds; ph, phloem; pp, palisade parenchyma; sp, spongy parenchyma; st, stomata; vb, vascular bundle; xy, xylem]. Scale bar: a = 50 µm; c = 500 µm; f = 200 µm; a, d, e = 50 µm; b = 10 µm.

The petiole, in cross section, is heart shaped. A large amount of non-glandular trichomes covers the single-layered epidermis (Fig. 3a, b). The cuticle and the content of the secretory cavities (Fig. 3c) reacted with Sudan III (Fig. 3b). Several druses (Fig. 3c) and starch grains were scattered in the ground parenchyma. These grains reacted with iodine solution (Fig. 3e).

The vascular system of petiole consists of a bicollateral vascular bundle in an open arch with invaginated ends (Fig. 3a, d). The reaction of the xylem with phloroglucinol/HCl is highlighted in Figure 3d. Phenolic compounds can be observed in the phloem, evidenced by the positive reaction with ferric chloride in the histochemical test (Fig. 3f).

Figure 3 – Anatomy of the petiole of Myrcia hatschbachii

Source: Authors (2018)

In where: [a, b, d-f: light microscopy; c: SEM]. Cross section. Petiole is heart shaped (a); Cuticle in reaction with Sudan III (b); Druses in the ground parenchyma (c); Xylem in reaction with phloroglucinol/HCl (d); Starch grains in reaction with iodine solution (e); Phenolic compounds in reaction with ferric chloride (f). [ct, cuticle; dr, druse; ep, epidermis; fi, fiber; gp, ground parenchyma; nt, non-glandular trichome; pc, phenolic compound; ph, phloem; st, stomata; xy, xylem]. Scale bar: a = 500 µm; b = 300 µm; d-f = 50 µm; c = 5 µm.

The stem of M. hatschbachii is oval in cross-section (Fig. 4a, b). The epidermis is uniserial and covered by a striated cuticle that reacts positively with Sudan III (Fig. 4c). Unicellular, non-glandular trichomes are frequently present (Fig. 4b).

Secretory cavities of stem are present in the cortex (Fig. 4a, c). The lipophilic content of the cavities reacted with Sudam III in the histochemical tests. The vascular system is bicollateral, surrounded by a discontinuous sclerenchymatic sheath that reacts positively in the hydrochloric phloroglucinol/HCl test (Fig. 4d). Phenolic compounds are present in the phloem (Fig. 4f), and lignified cells in the pith (Fig. 4e), as well as numerous calcium oxalate druses (Fig. 4g).

Figure 4 – Anatomy of the stem of Myrcia hatschbachii

Source: authors, 2018

In where: [a, c-f: light microscopy; b, g: SEM]. Cross section. Stem is oval and non-glandular trichomes are present (a, b); Cuticle in reaction with Sudan III (c); Fibers, xylem (d) and cells in the pith (e) in reaction with phloroglucinol/HCl; Phenolic compounds in reaction with ferric chloride (f); Calcium oxalate druses (g). [ct, cuticle; cv, secretory cavities; cx, cortex; dr, druse; ep, epidermis; fi, fiber; nt, non-glandular trichome; pc, phenolic compound; ph, phloem; pi, pith; xy, xylem]. Scale bar: a, b = 500 µm; d = 200 µm; c, e, f = 50 µm; g = 5 µm.

4 Discussion

It is important to highlight that this is the first report of the micromorphological, histochemical and anatomical characteristics of M. hatschbachii. Table 1 shows a summary of the anatomical characteristics of M. hatschbachii leaves compared to other species of the genus.

Table 1 – Comparative anatomical characters of leaves of species from genus Myrcia

Leaf anatomical characters	M. hatschbachii	M. guianensis1*	M. multiflora*	M. multiflora*	M. decrescens1*	M. cordiifolia	M. torta1*
		(JORGE et al., 2000)	(JORGE et al., 2000)	(DONATO; MORRETES, 2011)	(GOMES et al., 2009)	(GOMES et al., 2009)	(GOMES et al., 2009)
Epidermal cell walls	Wavy anticlinal	Wavy anticlinal	Straight anticlinal	Straight anticlinal	Wavy anticlinal	Wavy anticlinal	Wavy anticlinal
Mesophyll	Dorsiventral	Dorsiventral	Dorsiventral	Dorsiventral	Dorsiventral	Dorsiventral	Dorsiventral
Stomata	Paracytic	Ranunculaceous	Ranunculaceous	Paracytic	Paracytic	Paracytic	Paracytic
Trichomes	Unicellular	Unicellular	Unicellular	Unicellular	Unicellular	Unicellular	Unicellular
Palisade parenchyma	One layer	One layer	Three layers	Three layers	One layer	One layer	Three layers
Ground parenchyma	Secretory cavities	-	-	Secretory cavities	Secretory cavities	Secretory cavities	Secretory cavities
	Starch grains	-	-	Starch grains	-	-	-
	Druses**	Druses**	Druses**	Druses**	-	-	-
Midrib shape	Concave-convex	Concave-convex	Flat-convex	Concave-convex	Biconvex	Flat-convex	Flat-convex
Vascular system	C-shaped	Open arc	Open arc	-	Open arc	Open arc	Open arc

Source: Authors (2020)

In where: 1 Myrcia guianensis (Aubl.) DC. = Myrcia decrescens (O. Berg) Mattos = Myrcia torta DC. *Group: “pedra-hume-caá”, “pedra-ume-caá” or “insulin plant” (Cascaes et al., 2015). **Calcium oxalate druses.

Some features found in M. hatschbachii were also observed in other species belong to the genus Myrcia (JORGE et al., 2000; Retamales; Scharaschkin, 2015) or the family Myrtaceae (Metcalfe; Chalk, 1979; Landrum; Kawasaki, 1997; Wilson, 2011). For instance, in the leaves, wavy anticlinal epidermal cell walls, unicellular non-glandular trichomes, dorsiventral mesophyll and secretory cavities are common characteristics and these features contribute to the taxonomy of the genus and family.

According to Metcalfe and Chalk (1979), calcium oxalate crystals of various shapes are commonly found in Myrtaceae. Among the most common crystal are druses, which are common in several genera of Myrtaceae, such as Eucalyptus L’Hér, Eugenia, Gomidesia O. Berg, Psidium L., and Myrcia, and other genera from South America (Cardoso et al., 2009; Gomes et al., 2009; Saulle et al., 2018; Migacz et al., 2018). However, prismatic calcium oxalate crystals are also found in the family (SAULLE et al., 2018; MIGACZ et al., 2018), not only in the mesophyll and ground parenchyma, but also on the epidermis of the leaves (MIGACZ et al., 2018).

Myrcia multiflora contains prismatic crystals in the vascular bundles (DONATO; MORRETES, 2011). Crystals are scarce in M. guianensis (JORGE et al., 2000), whereas they are absent in M. coriacea (Sugden, 1985). Although the size and the amount of crystals vary among different taxa, their shape and location within a taxon is usually specific and may be considered as a taxonomic characteristic (Franceschi; Nakata, 2005). In the present study, several calcium oxalate druses are found in the spongy parenchyma of M. hatschbachii. This feature help in the diagnosis of the species.

The midrib of leaves has a concave-convex shape. A slightly concave-convex shape has been observed in M. multiflora (DONATO; MORRETES, 2011), a biconvex shape in M. decrescens, and a flat-convex shape in M. cordiifolia DC. and M. torta DC. (GOMES et al., 2009). The bicollateral vascular system, with a single C-shaped vascular bundle was found in M. hatschbachii and it is common in other species of Myrtaceae family (GOMES et al., 2009; DONATO; MORRETES, 2011). However, M. guianensis, M. multiflora (JORGE et al., 2000), M. cordiifolia, M. decrescens, and M. torta (GOMES et al., 2009) have an open arc vascular system.

Although they present variable forms of the midrib and the vascular system (DONATO; MORRETES, 2011; GOMES et al., 2009; JORGE et al., 2000), the species M. multiflora, M. guianensis or M. decrescens, and M. hatschbachii are native to Brazil and have geographic regions (Santa Catarina and Rio Grande do Sul) and phytogeographic domain (Atlantic Forest) in common (BFG, 2018). Thus, the anatomical diagnostic features contribute to the identification of these species and can assist further studies on the possible differentiation from the species of the genus Myrcia, including the insulin plants group, such as M. multiflora.

The vascular system of petiole consists of a bicollateral vascular bundle in an open arch with invaginated ends. Different outline was found in Myrcia multiflora, flat-convex shape with one vascular bundle in an open arc (DONATO; MORRETES, 2011). The shape and the pattern of the vascular system of the midrib and petiole are anatomical markers that can help to the identification of the species, as stated by some authors (Wosch et al., 2015; Bobek et al., 2016; Budel et al., 2018) and observed in the present study.

Phenolic compounds were found in great amount, through histochemical tests, in leaves, petiole, and stem. These data corroborate the main metabolites found in the family and genus. The family Myrtaceae (RODRIGUES et al., 2016) and the genus Myrcia (CASCAES et al. 2015) are characterized by the presence of phenolic compounds, in addition, the species M. hatschbachii presented a high content of these compounds in in vitro tests (GATTO et al., 2020).

5 Conclusion

The present study is the first to explore and document the anatomical and histochemical features of M. hatschbachii. Although most of the characteristics highlighted in the present study are common in the family Myrtaceae, the following anatomical and histochemical features taken together can help the identification of M. hatschbachii: druses in the spongy parenchyma, concave-convex midrib, heart-shaped petiole, oval-shaped stem, and C-shaped vascular bundle in the midrib and open arc shape with invaginated ends in the petiole; phenolic compounds in the phloem and lignified cells in the pith. Studies like this are required to assist informed decisions on the classification and taxonomy of the high diversified genus Myrcia as well as the family Myrtaceae. Further studies that integrate anatomical and histochemical characteristics with phylogenetic analysis are essential in understanding the evolution and ecological implications of the traits evidenced.

Acknowledgments

This work was carried out with the support of the Coordination of Improvement of Higher Education Personnel - Brazil (CAPES) - Financing Code 001. The authors are grateful to UEPG and c-Labmu for the assistance in the analysis, and to the taxonomist José Tadeu Weidlich Motta for the identification of the species.

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Authorship contributions

1 - Larissa Junqueira Gatto

Contribuition: Conceptualization, Methodology, Writing – original draft

2 - Vanessa Barbosa Bobek

Contribuition: Methodology, Writing – original draft

3 - Jane Manfron Budel

Contribution Writing – review & editing

4 - Natasha Tiemi Fabri

Contribuition: Methodology

5 - Josiane de Fatima Gaspari Dias

Contribuition: Methodology

6 - Obdulio Gomes Miguel

Contribuition: Supervision

7 - Marilis Dallarmi Miguel

Contribuition: Supervision