Copaifera appendiculata M. J. Silva, 2022

Silva, Marcos José Da & Santos, Igor Soares Dos, 2022, A lovely new, and potentially medicinal, species of Copaifera (Detarioideae, Fabaceae) from the Brazilian Cerrado supported by anatomical and morphological data, Phytotaxa 552 (2), pp. 127-150 : 129-146

Copaifera appendiculata M. J. Silva , sp. nov.

Type:— BRAZIL. Goiás: Niquelândia, Reserva Particular de Desenvolvimento Sustentável Legado Verdes do Cerrado , Núcleo Engenho , Área do Acaba Vida , ca. 1 km da porteira, lado esquerdo da estrada, 753 m. a.s.l., 14°41’59”S, 48°25’25”W, 26 January 2020, fl., M. J. Silva, I. S. Santos & B. S. Pereira 10631 (holotype UFG!; isotypes: CEN!, UB!) ( Figs. 1 , 2 ) .

Diagnosis:—This new species is characterized by having a dwarf, shrubby habit, leaves sessile or with petiole up to 1.8 cm long, and rachis with a conspicuous spiny prolongation 0.9–1 cm long, 2–4 (–5) pairs of leaflets, similar in size, coriaceous, glabrous or indumented at least on the midrib on the abaxial surface, with margins planar, translucent points conspicuous or inconspicuous, with one gland at the base, panicles smaller or slightly larger than the underlying leaves; flowers sessile, the sepals conspicuosly indumented on both surfaces, ovary evenly indumented, fruits glabrous, and seeds with an orange aril.

Description:—Shrubs (0.6–) 1–1.7 m tall; adult branches striated, glabrous and cinereous, with conspicuous scars, smooth or longitudinally cracked, young branches shortly tomentose, the trichomes hyaline, rusty, or ocher. Leaves 8.3–15 cm long; stipules 1.3–2.3 × 0.2–0.3 cm, falcate, persistent or tardily caducous, glabrous or glabrescent on the external surface; petiole 0.2–1.8 cm long; rachis 5–9 cm long, both sulcate above, striated, shortly tomentose, rusty or ocher, the rachis with an spiny prolongation 0.9–1 cm long; petiolule 1–3 mm long; leaflets (2)3–4(5) pairs, the proximal similar to the distal, or slightly smaller in size, thin or thick coriaceous, oblong, oblong-elliptic, ellipticfalcate, elliptic or ovate; opposite, or less often subopposite; 5–10 × 2.8–5.8 cm, glandular-punctate, the punctations translucent, visible or not; margins flat, ciliate, non-cartilaginous, with 1 nectary on each side immediately at the base; apex obtuse, slightly emarginate, non-mucronulate, base obtuse or slightly oblique, adaxial surface glabrous or sparsely puberulous, abaxial surface similar to the adaxial, but with trichomes shortly tomentose on the midrib; venation brochidodromous, midrib slightly shifted to the right side of the blade, prominent on the abaxial surface, secondary veins impressed on both surfaces. Panicles 13–19.5 cm long, smaller or slightly larger than the undelying leaves, lax, axillary; peduncle 1–2 cm long, rachis 12.5–17.5 cm long, secondary axes (6–) 9–11 cm long, all tomentoserusty, thickened by galls; bracts 3.2–3.3 × 2.9–3 mm, widely ovate, margins entire, ciliate, shortly villose externally; bracteoles 2.8–2.9 × ca. 1.9 mm, similar to the bracts; floral bud 4.7–4.7 × 3–3.1 mm, ellipsoidal, shortly tomentose. Flowers sessile, sepals 4, 5–5.1 × 2.2–2.3 mm, lanceolate, oval-lanceolate, or elliptic, densely sericeous internally, sparsely or densely sericeous externally, with conspicuous secretory cavities, margins entire, apex acute; stamens 10, filaments 6–8 mm long, glabrous; anthers 1.2–1.5 mm long, ellipsoidal, mucronulate, longitudinally dehiscent; ovary 2–.3 × 1.4–1.5 mm, ellipsoidal, densely villous throughout, the trichomes rusty; styles 3–5 mm long. Pod 2.6–3.6 × 2.2–2.5 cm, ellipsoidal, orbicular or obovoid, base asymmetric-attenuated, apex asymmetrically obtuse and mucronate, glabrous throughout, yellowish-green or orange-green when immature, reddish when ripe; seeds 1.5–2.8 × 1.3–1.7 cm, orbicular or elliptic-orbicular, black, aril orange to the middle region of the seed.

Additional specimens examined (paratypes): — BRAZIL. Goiás, Niquelândia, RPDS Legado Verdes do Cerrado, Núcleo Engenho, Área do Acaba Vida , ca. 200 metros antes da porteira, cerrado ralo, 771 m. a.s.l. 14°42’15”S, 48°25’24”W, 21 July 2019, fr., M. J. Silva 9878 ( UFG) , 9879 ( UFG); ib., ca. 1 km da porteira, lado esquerdo da estrada, 753 m. a.s.l., 14°41’59”S, 48°25’25”W, 26 January 2020, fl., M. J. Silva 10630 ( UFG), 10631 ( UFG) ; ibd., ca. 120 metros antes da porteira, 26 January 2020, fl., M. J. Silva 10642 ( UFG) ; ibd., cerrado ralo após morro, cerca de 750 metros a partir da estrada, 753 m. a.s.l., 14°41’59”S, 48°25’25”W, 26 January 2020, fl, M. J. Silva et al. 10638 ( UFG) , ibd. ca. 800 metros antes da porteira, ca. 60 metros a partir da estrada, 753 m. a.s.l., 14°41’59”S, 48°25’25”W, 26 January 2020, fl, M. J. Silva et al. 10645 ( UFG) ; ibd., ca. 600 metros antes da porteira 200 metros a partir da lateral esquerda, 752 m ASL, 14°41’59,61”S, 48°25’27,86”W, 26 January 2021, fl., M. J. Silva 11583 ( UFG), 11584 ( UFG), 11585 ( UFG) ; Área Capão do Bandeira , 693 m. a.s.l., 14°39’22”S, 48°29’35”W, 29 June 2019, fr., M. J. Silva 9667 ( UFG) ; Área da Filipa, morro após o ponto de apoio da área da Filipa , 705 m. a.s.l., 14°35’21”S, 48°26’05”W, 21 February 2020, fl., fr., M. J. Silva et al. 10974 ( UFG), 10975 ( UFG) ; Área do Poço Rosado , 100 m após o córrego, 14°36’42”S, 48°30’12”W, 623 m. a.s.l., 22 April 2021, fr., M. J. Silva et al. 12334 ( UFG) ; ibd., 90 metros a partir do córrego, 22 April 2021, fr., M. J. Silva et al. 12337 ( UFG) ; ibd., ca. 200 metros a partir do córrego, 619 m. a.s.l., 14°36’41”S, 48°30’14”W, 22 April 2021, fr., M. J. Silva et al. 12339 ( UFG) ; ibd., ca. 150 m, após o córrego, 22 April 2021, fr., M. J. Silva et al. 12340 ( UFG), 12341 ( UFG), 12342 ( UFG), 12342 ( UFG), 12343 ( UFG) ; Área da Trilha do Campo Cerrado , 656 m. a.s.l., 14°36’52”S, 48°29’34”W, 29 May 2021, fr., M. J. Silva 12578 (UFG), 12579 (UFG) ; ibd., ca. 180 metros a partir do córrego, M. J. Silva 12581 ( UFG), 12582 ( UFG), 12585 ( UFG); Área do Lama Preta, cerca de 400 metros a partir do 4º Córrego do Lama Preta , lado direito da rodagem, 27 January 2021, fl., M. J. Silva 11635 ( UFG), 11636 ( UFG), 11637 ( UFG), 11638 ( UFG), 11639 ( UFG), 11640 ( UFG), 11641 ( UFG), 11643 ( UFG) , ibd., 718 m. a.s.l., 14°39’36”S, 48°25’49”W, 23 June 2021, fr., M. J. Silva 12717 ( UFG), 12718 ( UFG), 12719 ( UFG), 12720 ( UFG) ; Área do Mirante, ca. 400 metros a leste do Mirante , 14°39’45”S, 48°27’31”W, 724 m. a.s.l., 25 January 2020, fl., M. J. Silva 10585 ( UFG), 10586 ( UFG) ; Área do Pasto do Romão, 100 metros a partir da estrada que leva ao pasto do Romão , 647 m. a.s.l., 14°37’07”S, 48°27’05”W, 27 January 2021, fl., M. J. Silva 11668 ( UFG), 11669 ( UFG), 11670 ( UFG), 11671 ( UFG) ; ibd., ca. 200 metros a partir da porteira do pasto do Romão , 656 m. a.s.l., 14°37’08”S, 48°27’06”W, 27 January 2021, fl., M. J. Silva 11679 ( UFG), 11680 ( UFG), 11681 ( UFG) ; ibd., cerrado ralo ca. 150 m após a mata, 24 Jun 2021, fr., M. J. Silva 12796 ( UFG), 12797 ( UFG), 12798 ( UFG), 12799 ( UFG) ; Área do Rio Traíras, ca. 100 metros antes de chegar à Ponte do rio Traíras , cerrado típico na lateral esquerda da estrada, 630 m. a.s.l., 14°36’34”S, 48°28’38”W, 25 January 2020, fl., M. J. Silva 10536 ( UFG) ; ibd., ca. 250 metros antes da ponte do Traíras, na lateral esquerda da trilha do Traíras , 668 m. a.s.l., 14°365’37”S, 48°28’54”W, 25 January 2020, fl., M. J. Silva 10539 ( UFG) ; ib., ca. 400 metros após a ponte do rio Traíras , cerca de 350 metros para dentro do Cerrado típico do lado esquerdo da estrada, 25 January 2020, fl., M. J. Silva 10558 (UFG), 10560 (UFG) .

Distribution and habitat:— Copaifera appendiculata has been encountered so far in the Legado Verdes do Cerrado Private Sustainable Development Reserve, in the municipality of Niquelândia, Goiás State, Brazil ( Fig. 4 ). Grows in “cerrado típicos”, “cerrados ralos”, “campos sujos”, and in the transition from “campos sujos” to “cerrados ralos”, on sandy-clay, litholic, and stony-clayey soils in level areas, slopes, or hilltops between 618 and 753 m. a.s.l.

Phenology:—Species collected with flowers in January and fruits in April, June and July.

Etymology:—The specific epithet “appendiculata” refers to the leaf rachis of the new species with a conspicuous terminal appendix, a character not mentioned in the specialized literature for the genus.

Vernacular name:—“Pau-de-óleo mirim” and “copaibinha” according to the employees at the Legado Verdes do Cerrado Sustainable Development Private Reserve.

Proposed conservation status:—The conservation status of Copaifera appendiculata was classified as Critically Endangered [CR, B1b (i, ii, iii, iv, v), as it is known from fewer than 10 localities and has an Extent of Occurrence of ca. 75.42 km 2. Additionally, most populations of this species have less than 20 individuals, and although they grow in an area protected by law, some sites demonstrate anthropic disturbances caused by the removal of the native vegetation and agricultural activities.

Notes:— Copaifera appendiculata is easily recognized as a shrub 0.6 to 1.7 m tall, leaves commonly with 2–3(4) pairs of leaflets, oblong, oblong-elliptic, elliptic-falcate or elliptic, the proximal leaflets similar to distal ones in size, with coriaceous blades, nerviform and ciliate margins, glabrescent adaxial surface, glabrescent abaxial surface, or slightly tomentose at least in the midrib, petioles and rachis striated, glabrescent or tomentose, the trichomes yellowish, rusty, or cinereous, the rachis with a spiny prolongation; stipules evident, 1.3–2.3 cm long, panicles usually smaller than the underlying leaves, flowers sessile with lanceolate, oval-lanceolate or elliptic sepals, densely sericeous internally, sparsely to densely sericeous externally, with conspicuous secretory cavities, in addition to ellipsoidal or orbicular fruits with asymmetrically attenuated bases and seeds with an orange aril.Among the subshrubby or shrubby species of Copaifera present in the Cerrado, C. marginata Benth. ( Figs. 3a–e ) is most similar to C. appendiculata , as both share a shrubby habit, leaves with 2 to 4 pairs of coriaceous leaflets, similar in size and poorly differentiated on both surfaces, in addition to the rachis and petiole indumented, flowers sessile with sepals indumented on both surfaces. C. appendiculata can be distinguished, however, by the leaf rachis having a spiny prolongation (vs. absent in C. marginata ), leaflets with or without visible translucent points, margins nerviform, flat, and usually ciliated (vs. without visible translucent points, cartilaginous, revolute, and with non-ciliated margins), panicles smaller or slightly larger than the underlying leaves (vs. two to three times larger than the underlying leaves), ovary ellipsoidal, densely villous throughout (vs. ovary pubescent on margins), fruits brown with margins glabrous (vs. purplish or reddish, tomentose or pubescent), and seeds orbicular or elliptic-orbicular with orange aril (vs. oblong with white aril)

Copaifera is a complex genus, and its dwarf Cerrado species are consistently mistakenly identified in herbarium collections (e.g., CEN, UB, IBGE), and the literature concerning the genus dealing with species from the Cerrado biome (e.g., Costa 2007, Souza et al. 2016) provide poorly informative descriptions and keys often have overlapping characteristics. As a result, other subshrubby or shrubby Copaifera species (such as C. elliptica , C. magnifolia Dwyer , C. malmei Harms , and C. nana Rizzini ) can easily be confused with C. appendiculata . All of those other species, however, have a foliar rachis without any prolongation (vs. prolonged in C. appendiculata ) and seeds with white arils (vs. orange), in addition to each being differentiated by other important characters (See Costa 2007, Martins-da-Silva et al. 2008, Souza et al. 2016), as shown in the table 1.

Leaf anatomical data:— Copaifera appendiculata has petioles with cordate contours in cross section ( Fig. 5a ), while C. marginata Benth. has petioles with plano-convex contours ( Fig. 5h ); both species have petioles covered by a thick cuticle, an unistratified epidermis with rectangular, quadrangular or, less frequently, rounded common cells with thickened walls ( Fig. 5b, c, i, j ); in C. marginata the cuticle is thicker and the common epidermal cells contain phenolic compounds ( Fig. 5i ). The latter species exhibits an unistratified hypodermis with rounded cells with thickened walls ( Fig. 5i, k ). The cortex of the species studied here comprises 1–3 layers of angular collenchyma cells ( Fig. 5b, k ), secretory cavities with wide lumens, and juxtaposed, elliptic and rounded secretory epithelial cells with thin peri- and anticlinal walls, diffusely distributed between the parenchyma cells ( Fig. 5c, f, g, i, n ). Those cells are responsible for the synthesis and secretion of different classes of secondary metabolites and substances with lipophilic natures into the lumens of the secretory cavities ( Fig. 5k ). Both species have a vascular system composed of collateral bundles, although C. appendiculata has a main vascular bundle in a closed arch, and 10 accessory vascular bundles, five on each side ( Fig. 5a ), while C. marginata has only one central vascular bundle ( Fig. 5h ). The vascular bundles of both species are surrounded by gelatinous and libriform fibers; in C. marginata they are interspersed between the fibers and diffused by the parenchyma occur sclereids ( Fig. 5i, k, m ). In both taxa, the large-caliber vascular bundle contains pith with parenchyma cells and secretory cavities ( Fig. 5f ). Non-glandular trichomes are found in both species, being more common in C. marginata ( Fig. 5j ), which also has polyhedral crystals associated with the fibers ( Fig. 5k ).

Rodrigues et al. (2011) and Carvalho et al. (2019) presented only one cross-sectional image of the petioles of both C. sabulicola J.A.S. Costa & L.P. Queiroz (2007: 394) and C. langsdorffii , showing an unistratified epidermis with a thick cuticle, the cortex with secretory cavities, and the vascular system comprising a single central vascular bundle and accessory vascular bundles in adaxial positions as in C. appendiculata and C. marginata . Copaifera sabulicola , however, has a petiole without a hypodermis (vs. present in C. marginata and absent in C. appendiculata and C. langsdorffii ), a vascular system with two accessory vascular bundles (vs. absent in C. marginata , 10 in C. appendiculata , and two in C. langsdorffii ), and a plano-convex contour (vs. cordate in C. appendiculata and planoconvex in C. marginata and C. langsdorffii ). The anatomical features of the petiole observed in the studied species are similar to those of members of the Leguminosae Juss. subfam. Detarioideae Burmeist. , tribe Detarieae DC. , which belongs to the genus Copaifera , as observed in the genus Hymenaea Linnaeus (1753: 1192) by Silva et al. (2012).

The rachis of Copaifera appendiculata has an ovoid contour, canaliculate, and slightly ondulate on the adaxial surface ( Fig. 6a ), while in C. marginata the rachis is evenly rounded and without undulations ( Fig. 6f ). The anatomy of the rachis of both species is very similar to that observed in the petiole, that is, C. marginada has a hypodermis as well as accessory vascular bundles and secretory cavities diffused throughout the parenchyma ( Fig. 6f, g ). In both taxa, the epidermis is unistratified, commonly with rectangular or quadrangular cells surrounded by a thick cuticle ( Fig. 6c, g ); the cortex comprises 2–4 layers of angular collenchyma, 3 to 7 layers of parenchyma, with some sclereids and secretory cavities dispersed throughout the parenchyma ( Fig. 6b, c, e, g, j ), and associated with fibers in C. marginata ( Fig. 6h ).

The vascular systems of the rachis of the species studied here are formed by collateral vascular bundles with a conspicuous sheath of fibers, which are associated with sclereids in C. marginata ( Fig. 6a, f, h ). C. appendiculata has a central vascular bundle in a closed arc, and four accessory vascular bundles facing the adaxial surface ( Fig. 6a, b ); C. marginata has only a central vascular bundle in a closed arc, which occupies the largest extension between the rachis tissues ( Fig. 6f, i ). The pith is conspicuous in these species, with parenchymatic cells and secretory cavities in C. appendiculata ( Fig. 6a, d ) but few parenchyma cells in C. marginata ( Fig. 6f ). Non-glandular trichomes were also observed in both species ( Fig. 6a, g, f ). This work provides the first descriptive report of rachis anatomy for Copaifera .

The midribs of the leaflets of Copaifera appendiculata have concave-convex contours; in C marginata they are plano-convex, with the first convexity angle being more conspicuous and acute ( Fig. 7a, f ). The epidermis of the species is unistratified, with common cells of varying sizes and shapes between the adaxial and abaxial surfaces. The epidermal cells of C. appendiculata are rectangular on the adaxial surface and ovoid on the abaxial surface (vs. quadrangular and rounded in C. marginata ), with the cells on the abaxial surface being larger than those on the adaxial surface ( Fig. 7b, c ); in contrast, the adaxial cells of C. marginata are larger than those on the abaxial surface ( Fig. 7g, h ). Stomata, distributed at the same level as the common cells of the adaxial epidermis, were observed only in C. appendiculata ( Fig. 7b ), whereas a hypodermis was found only in C. marginata ( Fig. 7g, h ). The cortex of both species comprises 2 or 3 layers of angular collenchyma ( Fig. 7c ) and 4 to 6 layers of parenchyma cells of varying shapes and sizes ( Fig. 7b, c, h, j ). Sclereids and secretory cavities were observed in both taxa ( Fig. 7c, g, h ). Polyhedral crystals were found near the fibers associated with the vascular system in C. appendiculata ( Fig. 7e ). The two species have a single collateral bundle in a closed arch with a conspicuous sheath consisting of gelatinous and libriform fibers ( Fig. 7d, e, g, i ), and pith composed of parenchymatic cells of varying dimensions ( Fig. 7d, i ).

Some of the anatomical characters observed in the midrib of the species studied here (e.g., a unistratified epidermis, parenchymatic pith, vascular system with collateral bundles [the largest of which are surrounded by a conspicuous fiber sheath], presence of collenchyma in a subepidermal position and secretory cavities diffused by the cortex) also occur in C. langsdorffii ( Oliveira et al. 2008; Rodrigues et al. 2011; Nascimento et al. 2014), in C. sabulicola ( Carvalho et al. 2019) , and in C. pubiflora Bentham ( Ferreira & Flores 2013). Other characters, however, support the taxonomic separation of the species studied here, and distinguish them from the two previously mentioned taxa, such, as a hypodermis present on the adaxial surface of C. marginata (vs. absent in the others), the presence of two accessory vascular bundles in C. sabulicola (vs. 3 or 4 in C. langsdorffii and absent in the others), and a biconvex contour in C. langsdorffii (vs. concave-convex in C. appendiculata , and plano-convex in C. marginata , C. sabulicola and C. pubiflora ).

The leaf blades of both species have a unistratified epidermis with rectangular, quadrangular or ovoid common cells covered by a thick cuticle ( Fig. 8a, b, g, h ). Both species have a dorsiventral mesophyll composed of three layers of palisade parenchyma, although C. appendiculata has 2 or 3 layers of spongy parenchyma (vs. 2 layers of palisade parenchyma and 3 or 4 layers of spongy parenchyma in C. marginata ). Both species also have large diffuse secretory cavities by the chlorenchyma ( Fig. 8a, d, g ), although they are more common in C. appendiculata , with polyhedral crystals close to the fibers associated with the vascular bundles ( Fig. 8f ). Both species have collateral vascular bundles of larger and smaller calibers in the mesophyll, the first ones surrounded by a sclerenchymatic sheath that projects to both surfaces of the leaf blade ( Fig. 8b, e, h, k ). The leaves of both species are hypostomatic, and their stomata have large substomatic chambers, distributed at the same level as the other common cells ( Fig. 8d, j ). An unistratified epidermis with juxtaposed cells with a thick cuticle, a dorsiventral mesophyll composed of up to 3 layers of palisade parenchyma, hypostomatic leaves, secretory cavities, and collateral vascular bundles with fiber sheaths projected to both sides of the epidermis in the leaf blade, appear to be common characters in the genus Copaifera , as described for C. sabulicola by Carvalho et al. (2019), for C. langsdorffii by Nascimento et al. (2014) and Melo Júnior et al. (2012), and for C. pubiflora by Ferreira & Flores (2013), and as described for members of the Hymenaea clade (also belonging to Detarioideae subfam., and sister of the Copaifera clade) ( LPWG 2017; Pinto et al. 2018).

The numbers of palisade parenchyma layers differentiate C. appendiculata from C. marginata , and both of them from C. sabulicola and C. langsdorffii , which have, respectively, 3, 2, 2 or 3, and 1 or 2 layers (this study, Oliveira et al. 2008; Rodrigues et al. 2011; Nascimento et al. 2014; Carvalho et al. 2019). The leaf margins of C. appendiculata are obtuse, and comprise, in a subepidermal position, a layer of angular collenchyma, 2 layers of parenchyma cells, and a marginal collateral vascular bundle surrounded by a sclerenchymatic sheath composed of up to 4 layers of cells ( Fig. 8a ). C. marginata , however, has a conspicuously rounded leaf edge, with two layers of parenchyma and secretory cavities, in addition to a collateral vascular bundle sheath with fibers having 7 to 10 cell layers ( Fig. 8g ). The xylem vessel elements in the vascular bundles of C. appendiculata are arranged in straight lines ( Fig. 8a ), but form arches in the congenera ( Fig. 8g ). The leaf margin of the two species studied here have vascular bundles with conspicuous layers of fibers (similar to those of C. sabulicola and C. langsdorffii ), although they are more expressive in C. marginata ( Fig. 8g ).

Some of the anatomical characters observed in the leaf tissues of the studied species, such as a thick cuticle, a mesophyll with more than two layers of palisade parenchyma, and hypostomatic leaves, reflect the habitat and environmental conditions of the Cerrado biome where they were collected, with its two well-defined seasons with strong solar radiation and luminosity, and often with water scarcities and excessive winds ( Cole 1986, Rossatto et al. 2015). A thick cuticle under those conditions allows the better reflection of sunlight, minimizes excessive water losses through transpiration, and provides the leaves with protection against mechanical stress (Pyyko 1966, Dickison 2000, Alquimi et al. 2006, Evert 2006, Nascimento et al. 2014, Carvalho et al. 2019). A palisade parenchyma with more than one layer of cells likewise makes photosynthetic processes more efficient, as it acts as a light guide, propagating light rays through tubular vacuoles and intercellular spaces ( DeLucia et al. 1996, Silva et al. 2012, Ramos et al. 2015, Carvalho et al. 2019). The thick-walled fiber sheaths in the vascular bundles reach both surfaces of the epidermis and act to prevent the collapse of the underlying tissues during periods of drought, serve as protective sheaths for the bundles, and aid water flow from the xylem to the mesophyll and epidermis ( Pizzolato et al. 1976, Metcalfe & Chalk 1950, 1983, Monteiro et al. 1985, Choong et al. 1992); the fibers envolving the vascular bundles in the midrib, petiole, and rachis provide mechanical support to those leaf structures (Dickson 2000), act to inhibit biotic attacks (ScreminDias 2006), and promote water retention (in the case of gelatinous fibers) ( Dickison 2000, Bieras & Sajo 2009), while the secretory cavities dispersed in the mesophyll act to protect against herbivory with defensive compounds, especially those of phenolic natures ( Fahn 1979, Dickison 2000). Those compounds also act as antioxidants ( Souza et al. 2007, Gengaihi et al. 2014).

The leaf anatomical characters described for Copaifera appendiculata and C. marginata are related to xeromorphism and are shared with other Cerrado species ( Morretes 1966, 1969, Bieras & Sajo 2009, Pinheiro et al. 2017). Xeromorphic leaf anatomical features are related to the evolutionary history of the Cerrado, as its first floristic elements have long faced water deficit conditions as well as soil acidity and toxicity during the climatic oscillations of the Tertiary and Quaternary eras ( Bieras & Sajo 2009). Those features were likely selected to ensure survival during the past periods of climatic adversity, but have been retained by the modern flora to protect against a variety of biotic and abiotic factors.

Histochemical data:—The histochemical tests performed on the leaf blades, petioles, rachises, and midribs of C. appendiculata and C. marginata were negative for proteins (by Coomassie Blue and Xylidine Ponceau) as well as for tannins (by vanillin-hydrochloric acid); they were positive, however, for the presence of lignin, alkaloids, reducing sugars, phenolic compounds, starch, and lipids. Lignin was found in the xylem vessel elements, fibers, and sclereids ( Fig. 9a–d and Fig. 10a–d ), and hypodermis ( Fig. 10d ). According to Silva et al. (2012) and Munk et al. (2015), lignin is a complex aromatic polymer that confers rigidity, mechanical support, and protection to internal tissues, aids water retention, and acts in the defense against both biotic (e.g., microorganisms) and abiotic agents by reinforcing the cell walls (Habermeht & Fliegner 1998, Dickison 2000, Firmino et al. 2006, Scatena & Scremin-Dias 2006). Lignins were likewise reported by Carvalho et al. (2019) in xylem cells and the fibers of C. sabulicola .

Alkaloids were found in the cortical and medullary parenchyma cells, in the xylem of the petiole ( Fig. 9e, f , Fig. 10e ) and rachis ( Fig. 10f ), inside the secretory cavities and epithelial cells ( Fig. 9g, h ), and in the palisade parenchyma ( Fig. 9i , Fig. 10g ) of both species. Alkaloids were likewise found in the secretory cavities, idioblasts, and mesophyll cells of C. langsdorffii and C. sabulicola by Rodrigues et al. (2011) and Carvalho et al. (2019) respectively, although they apparently are not present in all Copaifera species ( Gurgel 2009). Alkaloids are secondary metabolites composed of nitrogenous heterocyclic organic molecules, usually derived from amino acids. They act in plant defenses against pathogens and herbivores, and are used by human populations to treat gastrointestinal disorders and cardiovascular diseases; they also have analgesic, anti-inflammatory, antioxidant, antipyretic, antitumor, and immunomodulatory properties ( Sepúlveda-Jiménez et al. 2003, Brito et al. 2008, Ng et al. 2012, Li et al. 2017).

Reducing sugars were found in the petiole ( Fig. 9j ), rachis ( Fig. 9k ), and in the border region of the collenchyma ( Fig. 9l ) in C. appendiculata , and in the epithelial cells of the secretory structures of the leaf blade ( Fig. 10h ), associated with phloem and in parechimatic cells in the petiole ( Fig. 10i, j ), in the secretory cavities of the epithelial cells in the rachis ( Fig. 10k ), and in the palisade parenchyma ( Fig. 10l ) of C. marginata . Those sugars are derived from primary metabolism (photosynthesis) or from the hydrolysis of starch, and have been shown to be efficient agents against oxidative threats agents ( Lunáčková et al. 2000, Silva et al. 2003, Castellión et al. 2010).

Phenolic compounds were found in the common cells of the epidermis, parenchyma, and phloem and in secretory cavities, including in the epithelial cells ( Fig. 9 and 10m, n, o , Fig. 11a, c , Fig. 12a–d ) of both the species. Rodrigues et al. (2011) and Carvalho et al. (2019) in their studies of C. langsdorffii and C. sabulicola , also identified those substances in phloem cells and secretory cavities. Phenolic compounds are commonly found inside the vacuoles of idioblasts and the lumens of secretory cavities and ducts ( Carvalho et al. 2019), and are present in several species of Copaifera and others plants. They demonstrate antioxidant activities by minimizing damage caused to cells by free radicals (e.g., membrane peroxidation, DNA damage), and act in defenses against pathogens and herbivores ( Nascimento & Langenheim 1986, Souza et al. 2007, Gengaihi et al. 2014, Batista et al. 2016, Moudache et al. 2016).

Starch grains were found in the medullary parenchyma cells, and associated with the vascular bundles of the petiole and rachis, and the xylem of parenchyma rays of both species ( Fig. 11d, e , Fig. 12e, f ). Starch is a reserve carbohydrate composed of amylose and amylopectin, and is commonly found in plant reserves (e.g., tubers and roots, among others) and reproductive organs, and can be hydrolyzed to produce reducing sugars ( Lunáčková et al. 2000, Appezzato-da-Glória & Carmello-Guerreiro 2003, Jacomassi et al. 2010).

Lipids were abundant in the leaf tissues of the species studied here, especially in their secretory cavities ( Fig. 11f–l , Fig. 12g –l ). Tests using Nile Blue stain revealed that the secretions of lipophilic nature observed in the secretory cavities and epithelial cells are acidic lipids, and were found in all of the leaf specimens analyzed ( Fig. 11f–h , Fig. 12g –i ); lipids were also observed in the form of droplets in the palisade parenchyma cells of C appendiculata ( Fig. 11l ). The fatty nature of the cuticle covering the epidermal cells of both taxa was also evident ( Fig. 11i , 1 , Fig. 12k, l ), with Buarque et al. (2020) and Tiago et al. (2020) likewise reporting lipidic secretions in the secretory cavities, epithelial cells, and cuticles of C. langsdorffii and Hymenaea courbaril Linnaeus (1753: 1192) (taxa known for their medicinal properties). Buarque et al. 2020 performed histochemical tests and gathered ontogenetic and ultrastructural evidence to determine that the lipids were synthesized in the secretory cavities and in the chlorophyll parenchyma of their focal species. Fineschi & Loreto (2012) reported that lipid production in plants is constitutive, can be induced, and is related to defenses against biotic or abiotic stressors (e.g., attacks by insects and pathogens, oxidative stress, or heat shock). Acidic lipids can be found in the secretory structures of many different botanical families, such as Fabaceae , Anacardiaceae R.Br., And Asteraceae Martinov , among others ( Langenheim 1994, Sant’Anna-Santos et al. 2006, Zarinkamar et al. 2021). Those substances act as hydrogen ion carriers in oxidative phosphorylation mechanism ( Kocherginsky 2009) and can bind to cytotoxins ( Gasanov et al. 1997). Several authors (e.g., Nascimento & Langenheim 1986, Veiga-Júnior & Pinto 2002, Leandro et al. 2012) identified terpenes, especially sesquiterpenes and diterpenes, as constituents of secreted oils that have biological activities. Other authors (e.g., Basile et al. 1988, Carvalho et al. 2005; Gomes et al. 2010, Izumi et al. 2012; Leandro et al. 2012, Vargas et al. 2015, Carneiro et al. 2020) have reported that the terpenes found in Copaifera have pharmacological and therapeutic properties (e.g., analgesic, anti-inflammatory, and antitumor activities), and provide the plants with defenses against microorganisms.

Finally, we verified that the chemical compounds present in the leaf tissues of the species studied here are quite diverse (e.g., alkaloids, lipids, reducing sugars, and phenolic compounds), in agreement with many reports for the genus Copaifera – which reinforces the possibility of C. appendiculata and C. marginata as medicinal species – although additional phytochemical and biochemical studies will still be needed to better elucidate, isolate, and categorize their biologically active chemical constituents. In fact, a study of the chemical profiles of the leaves of other species of Copaifera from the Brazilian Cerrado (including C. appendiculata [as Copaifera sp. ] and C. marginata ) is currently being carried out. The study has already detected the presence of quinic acid and kaurenoic acid in C. marginata , of quercetin-3-O-L-rhamnoside in C. appendiculata , as well as sixteen isomers of galloylquinic acid - substances that have already been reported for other species of the genus (with confirmed medicinal potentials) (See Mota et al. 2017; Oliveira et al. 2020).

Figures

FIGURE 1. Copaifera appendiculata M. J. Silva: a flowering branch. b stipule. c distal portion of the rachis showing the spinescent extension. d–h variations in leaflet shapes. i trichomes on the abaxial surface. j trichomes on the adaxial surface. k detail of the leaflet margin and trichomes. l detail of the leaflet apex. m floral bud. n ventral surface of the bract. o detail of the bract margin. p flower, note the sepals conspicuously indumented ventrally. q ventral surface of the sepal. r detail of trichomes on the ventral surface of the sepal. s stamen. t ventral (left) and dorsal (right) surfaces of the anther. u gynoecium, note the uniformly indumented ovary. v detail of the trichomes on the ovary. w detail of the stigma. x fruit. y fruit after opening, note the twisted valve and prolonged hilum. z side view of the seed (drawn by Renato Cristiano Gualberto from the holotype).

FIGURE 2. Copaifera appendiculata M. J. Silva: a habit. b flowering branch. c portion of the inflorescence, note flowers being visited by ants. d detail of a raceme, showing floral buds and bracts. e flower in frontal view. f fruiting branch. g fruit after opening, note the twisted valve and prolonged hilum. h seed in side view, note the orange aril and black testa (Photographs by M. J. Silva).

FIGURE 3. Copaifera marginata Benth.: a habit, note its shrubby and cespitose aspect. b inflorescence. c portion of the inflorescence, note the light green elliptic buds. d fruiting branch, note the purplish or reddish fruits when mature. e imature seed, note the white aril. (Photographs by M. J. Silva).

FIGURE 4. Distribution of Copaifera appendiculata M. J. Silva: a map of Brazil, highlighting Goiás State in gray. b map of Goiás State, its limits, and elevational variations. c Municipality of Niquelândia, (dotted area) with species distribution points. Abbreviations for the Brazilian states (BA, Bahia; GO, Goiás; MG, Minas Gerais; MS, Mato Grosso do Sul; MT, Mato Grosso; TO, Tocantins) and the Federal District (DF).

FIGURE 5. Cross-section of the petioles of Copaifera appendiculata (a–g) and C. marginata (h–n): a, h general aspect of the petiole. b, i detail of the epidermis and internal tissues. c, j general aspect of the adaxial surface. d and k general aspects of the abaxial surface. e accessory vascular bundle. f, l detail of the larger vascular bundle. g, n detail of the secretory cavity. m detail of the fibers, phloem, and sclereids. (ab = accessory vascular bundle; ac = angular collenchyma; c = cuticle; fi = fibers; ep = epidermis; et = secretory epithelium; fi = fibers; hy = hypodermis; lu = lumen; pa = parenchyma; ph = phloem; me = pith; sc = sclereids; tr = trichomes; vb = vascular bundle; xy = xylem; * = phenolic compounds. arrowhead = prismatic crystals. * = secretory cavity). Scale: a, h = 500 μm; c, d, f, j, l = 200 μm; b, e, g, i, k, m, n = 50 μm.

FIGURE 6. Cross-section of the rachis of Copaifera appendiculata (a–e) and C. marginata (f–j): a, f general aspect of the rachis. b. detail of the acessory vascular bundles and adjacent tissues. c, g detail of the epidermis and hypodermis in g, parenchyma, collenchyma, fibers, sclereids, and secretory cavity in c. d, i. detail of the largest vascular bundle. e, j detail of the secretory cavity. h. detail of the fibers, phloem, sclereids, and secretory cavity. (ab = accessory vascular bundle; ac = angular collenchyma; c = cuticle; fi = fibers; ep = epidermis; et = secretory epithelium; fi = fibers; hy = hypodermis; lu = lumen; pa = parenchyma; ph = phloem; me = pith; sc = sclereids; tr = trichome; xy = xylem; * = secretory cavity. arrowhead = polyhedrical crystals). Scale: a, f = 500 μm; b, d, i = 200 μm; c, e, g, h, j = 50 μm.

FIGURE 7. Cross-sections of the midribs of Copaifera appendiculata M.J. Silva (a–e) and C. marginata (f–j): a, f. General aspect of the midrib. b, g detail of the epidermis, fibers, parenchyma, sclereids, and hypodermis of the adaxial surface. c, h detail of the epidermis, hypodermis, collenchyma in h, fibers, parenchyma, sclereids, and secretory cavity of the adaxial surface. d, i detail of the largest vascular bundle.e detail of the fibers. j detail of the secretory cavity (ac = angular collenchyma; et = secretory epithelium; fi = fibers; ep = epidermis; hy = hypodermis; lu = lumen; pa = parenchyma; ph = phloem; me = pith; sc = sclereids; xy = xylem; black arrowhead = stomata; white arrowhead = polyhedrical crystal. * = secretory cavity). Scale: a, f = 500 μm; d, i = 200 μm; b, c, e, g, h, j = 50 μm.

FIGURE 8. Cross sections of the leaf blades of Copaifera appendiculata M.J. Silva (a–f) and C. marginata (g–l): a, g general aspect of the margin. b, h general aspect of a leaf blade. c, i detail of the epidermis and palisade parenchyma (adaxial surface). d, j. detail of the epidermis, spongy and palisade parenchyma (abaxial surface). e, j, k detail of the epidermis, vascular bundle, secretory cavity in d, palisade and spongy parenchyma. e, k detail of a vascular bundle. f detail of a polyhedrical crystal. l. detail of a secretory cavity in the leaf margin. (ep = epidermis; et = secretory epithelium; fi = fibers; lu = lumen; ph = phloem; pp = palisade parenchyma; sp = spongy parenchyma; vb = vascular bundle; xy = xylem; black arrowhead = stomata; * = secretory cavity). Scale: a, b, g, h = 200 μm; c–e, i–l = 50 μm; f = 20 μm.

FIGURE 9. Cross-section of the leaf blade of Copaifera appendiculata M.J. Silva submitted to histochemical tests. a, b, e, f, j, n, o petiole. c, g, h, k, m rachis. d midrib. i leaf blade. l. margin. a–d lignin in the xylem vessel elements, fibers, and sclereids. e–i total alkaloids in the tissues indicated by arrows and *. j–l reducing sugars in the tissues indicated by arrows and *. m–o phenolic compounds in the tissues and inside the secretory cavity, indicated by arrows and *. (c = cuticle; fi = fibers; ep = epidermis; et = secretory epithelium; lu = lumen; ph = phloem; pp = palisade parenchyma; sc = sclereids; st = secretory cavity; sp = spongy parenchyma; vb = vascular bundle; xy = xylem). Scales: l, m = 200 µm; a–g, i, j, n, o = 50 µm; h, k = 20 µm.

FIGURE 10. Cross-section of a leaf blade of Copaifera marginata Benth. submitted to histochemical tests. a, e, i, n, o petiole. b, c, f, j, k, m rachis. d midrib. g, h, l leaf blade. a–d lignin in the xylem vessel elements, hypodermis cells, fibers, and sclereids. e–g total alkaloids indicated by arrows. h–l reducing sugars in tissues and in the epithelial cells of the secretory cavity, indicated by arrows. m–o phenolic compounds in the tissues and epithelial cells of the secretory cavity, indicated by *. (c = cuticle; fi = fibers; ep = epidermis; et = secretory epithelium; hy = hypodermis; lu = lumen; ph = phloem; pp = palisade parenchyma; sc = sclereids; st = secretory cavity; sp = spongy parenchyma; tr = trichome. xy = xylem). Scales: a, i, m = 200 µm. b-h, j, l, n = 50 µm; k, o = 20 µm.

FIGURE 11. Cross sections of leaf of the Copaifera appendiculata M.J. Silva submitted to histochemical tests. a midrib. b margin. c, d, f, i, j petiole. e, g, k rachis. h, l Leaf blade. a–c phenolic compounds in tissues and within the secretory cavity indicated by *. d, e starch grains in the parenchymatous pith and near the vascular system). f-h acidic lipids inside the secretory cavity indicated by *. i–l total lipids stained red-orange inside the secretory cavity and epithelial cells indicated by * and arrows. Lipid droplet indicated by arrow, and intense cuticle coloration in l. (c = cuticle; fi = fibers; ep = epidermis; et = secretory epithelium; lu = lumen; ph = phloem; pp = palisade parenchyma; st = secretory cavity; sp = spongy parenchyma; xy = xylem). Scales: a = 500 µm; b–e, h, i, k, l = 50 µm; f, g, j = 20 µm.

FIGURE 12. Cross-section of the leaf blade of Copaifera marginata Benth. submitted to histochemical tests. a, b midrib. c margin. d, i, j, l leaf blade. e, g, k petiole. f, h, j rachis. a–d. phenolic compounds in tissues and the epithelial cells of the secretory cavity. e, f starch grains in the parenchymatic pith and near the vascular system. g–i acidic lipids inside the secretory cavity and in the epithelial cells indicated by *. j–l total lipids stained red-orange inside the secretory cavity and epithelial cells indicated by arrows. Note the intense cuticle coloration in k, l. (c = cuticle; fi = fibers; ep = epidermis; et = secretory epithelium; hy = hypodermis; lu = lumen; ph = phloem; pp = palisade parenchyma; sc = sclereids; st = secretory cavity; sp = spongy parenchyma; xy = xylem). Scales: a = 500 µm; b–f, k, l = 50 µm; g–j = 20 µm.