Morphoanatomy of the Leaflets of the Hymenaea Clade (Fabaceae: Detarioideae) Reveals Their Potential for Taxonomic and Phylogenetic Studies

Botanical Journal of the Linnean Society, 2018, 187, 87–98. With 4 figures.

Morphoanatomy of the leaflets of the Hymenaea clade (Fabaceae: Detarioideae) reveals their potential for taxonomic and phylogenetic studies

RAFAEL B. PINTO1,2*, MAKELI G. LUSA2,3, VIDAL DE F. MANSANO4, Downloaded from https://academic.oup.com/botlinnean/article/187/1/87/4965893 by guest on 25 September 2021 ANA MARIA G. DE AZEVEDO TOZZI2 and JULIANA LISCHKA SAMPAIO MAYER2

1Universidade Federal de Goiás, Instituto de Ciências Biológicas, Departamento de Botânica, Programa de Pós-Graduação em Biodiversidade Vegetal, Campus Samambaia, CEP 74690-900, Goiânia, GO, Brazil 2Universidade Estadual de Campinas, Instituto de Biologia, Departamento de Biologia Vegetal, Programa de Pós-Graduação em Biologia Vegetal, Rua Monteiro Lobato 255, CEP 13083-862, Cidade Universitária Zeferino Vaz, Campinas, SP, Brazil 3Universidade Federal de Santa Catarina, Departamento de Botânica, CEP 88040–900, Campus Universitário Prof. João David Ferreira Lima, Bairro Trindade, Florianópolis, SC, Brazil 4Instituto de Pesquisa Jardim Botânico do Rio de Janeiro, DIPEQ, Rua Pacheco Leão, 915, CEP 22460- 030, Jardim Botânico, Rio de Janeiro, RJ, Brazil

Received 16 January 2017; revised 29 August 2017; accepted for publication 9 February 2018

The Hymenaea clade is part of the resin-producing clade of Fabaceae subfamily Detarioideae and is composed of three genera distributed mainly in tropical regions of Africa and South America. Anatomical characteristics of the leaflets are known for just a few representatives of this clade. This study aimed to improve this knowledge with morphoanatomical and histochemical characterizations of several species of Guibourtia, Hymenaea and Peltogyne. Our results support previous findings related to the presence of glandular cavities in Hymenaea and also add new evidence of the presence of these cavities in Guibourtia and Peltogyne. Absence of cavities was verified for H. velutina and P. catingae. Morphoanatomical characterization of the internerval region and histochemical analyses of the epidermis proved useful in delimiting species in the three genera. The position of the cavities in the leaflet lamina was also relevant in differentiating the three genera of this clade from other representatives of the subfamily. Our results contribute to the process of searching for features with phylogenetic signals and will be included in further phylogenetic analyses.

ADDITIONAL KEYWORDS: Detarieae – glands – jatobá – Leguminosae – morphology – phylogeny – resin – systematics.

INTRODUCTION and one in Africa; and Peltogyne Vogel with c. 21 Neotropical species. These genera share the tree habit The Hymenaea clade belongs to the recently recognized (although a few species of Hymenaea are predominately Fabaceae subfamily Detarioideae [Legume Phylogeny shrubs: H. eriogyne Benth., H. rubriflora Ducke and Working Group (LPWG), 2017], which has been H. travassii Kuhlmann ex Paes) and bifoliolate leaves, strongly supported as monophyletic (Fougère-Danezan, but they differ morphologically, mainly by the corolla Maumont & Bruneau, 2007; Bruneau et al., 2008). The (absent in Guibourtia), stamen length (heteromorphic clade is currently composed of three genera: Guibourtia in Peltogyne) and fruit dehiscence (indehiscent in Benn. with c. 15 species, two in the Neotropics and 13 in Hymenaea). Africa; Hymenaea L. with c. 16 species in the Neotropics The detarioids are known mainly for their resin production and the resin of several representatives, *Corresponding author. E-mail: [email protected] including the Hymenaea clade, is used in medicines,

© 2018 The Linnean Society of London, Botanical Journal of the Linnean Society, 2018, 187, 87–98 87 88 R. B. PINTO ET AL. jewelry and artefacts (Langenheim, 2003). Studies of The occurrence of mucilage in the vegetative organs ancient trading of artefacts indicate resins of Hymenaea is usually associated with carbohydrate storage, water as one of the main products of the commercial route storage and reduction of transpiration, protection from between East Africa and Arabia during the Middle Ages intensive radiation by light scattering or reflection, and (Regert, Devièse & Le Hô, 2008; Crowther et al., 2015). protection against herbivory (Gregory & Baas, 1989). In Brazil, its exploitation reached c. 85 tons per year The species of the three genera of the Hymenaea during the 1940s (Serviço de Estatística e Economia clade are distributed in different environments of the Brasileira, 1954). Amber from Hymenaea spp. is also tropics in Africa and South America. For example, associated with palaeontological research because it most Guibourtia spp. are distributed in humid forests occasionally preserves structures of plants or animals of the African continent, but some also occur in open (especially invertebrates) over millions of years due to vegetation of the South American continent (chaco Downloaded from https://academic.oup.com/botlinnean/article/187/1/87/4965893 by guest on 25 September 2021 its hardness and fossilization capability (Poinar, 1991; and Brazilian cerrado) and near the sea in Cuba Poinar & Brown, 2002; Calvillo-Canadell, Cevallos- (Léonard, 1949, 1950). Similarly, species of Hymenaea Ferriz & Rico-Arce, 2010). and Peltogyne are distributed mainly in ombrophilous The resins usually have an ecological function vegetation of South America (Amazonian and Atlantic in providing protection against dehydration and/ forests) and in open vegetation (e.g. cerrado and or herbivory, favouring colonization of areas with caatinga) (Lee & Langenheim, 1975; Silva, 1976). high insolation and high incidence of insects, such Besides the different geography, in each genus the as in tropical vegetation (Langenheim, 1994, 2003). species can vary regarding leaflet shape, thickness, Arrhenius & Langenheim (1983) also found a brightness, pubescence and colour; however, there is fungal growth inhibitory property when studying little knowledge of the anatomy of the leaves of this this effect in resins of Hymenaea and Copaifera group, especially from a comparative standpoint. In L. Structures associated with oil/resin exudation addition to this gap in the knowledge on morphology have also been explored in a taxonomic context in of the Hymenaea clade, Guibourtia is probably not several groups of plants, including Asteraceae (Lusa, monophyletic (R. B. Pinto et al., unpublished data). Costa & Glória, 2016a), Calophyllaceae (Caddah Thus, our goal was to indicate anatomical features et al., 2011), Malpighiaceae (Araújo & Meira, 2016) that could help to better delimit the taxa and also be and Velloziaceae (Mello-Silva, 1995). In Fabaceae, used as an indication of the lineages present in the secretory structures are distributed along different Hymenaea clade. lineages and have been taxonomically explored in some genera, such as Lonchocarpus Kunth (Teixeira, Castro & Tozzi, 2000), Copaifera (Cascon & Gilbert, MATERIAL AND METHODS 2000), Myrocarpus Allemão, Myroxylon L.f. and Myrospermum Jacq. (Sartori & Tozzi 2002), Zornia Samples of leaflets of adult plants from representatives J.F.Gmel. (Fortuna-Perez, Castro & Tozzi, 2012) of the Hymenaea clade were collected in several and genera from tribe Cercideae (Duarte-Almeida locations in Brazil and for some species fragments et al., 2015). Usually referred to as translucent dots from herbarium collections were also used. When in the leaflet lamina, the secretory cavities of the possible, different individuals from each taxon were leaflets have been used as a diagnostic character in observed and the most representative picture was identification keys (Polhill, 1981). selected. Table 1 includes the vouchers with more Resin secretory cavities have been studied in a precise locality information. few Hymenaea spp. by Langenheim et al. (1982), who Fragments taken from the middle third of the leaflet suggested that the number and the position of resin blade were fixed in Karnovsky’s solution (Karnovsky, pockets may have taxonomic value. However, the 1965; modified to incorporate phosphate buffer of pH authors attribute the organization of the cavities to 7.2), subjected to vacuum and dehydrated using ethyl ecological factors, such as herbivory pressure. Paiva alcohol at different concentrations. They were then & Machado (2007) studied the secretory cavities infiltrated with hydroxyethyl methacrylate plastic resin in leaflets of H. stigonocarpa Mart. ex Hayne and (Leica Historesin; Heraeus Kulzer, Hanau, Germany), concluded that, at least in this species, the cavities are according to the manufacturer’s instructions. organized randomly; thus, neither the number nor the The samples were sectioned to a thickness of 5–7 mm position of the cavities seems to have taxonomic value. using a rotary microtome (Model RM 2245; Leica Besides oil/resin, the presence of mucilage in the leaf Microsystems Nussloch GmbH, Nussloch, Germany), tissue restricted to idioblasts or accumulated between in transverse and longitudinal sections to observe the parenchymatous and epidermal cells of the epidermis structure of the glands. These materials were stained can also help in the systematics for genera of tribe with 0.05% toluidine blue in citrate-phosphate buffer, Crotalarieae (Boatwright, Tilney & Van Wyk, 2009). pH 4.5 (Sakai, 1973), and mounted in Entellan synthetic

Table 1. Species, general localities and vouchers of the material used in this study

Species Locality Voucher Herbarium

Guibourtia chodatiana (Hassl.) Campinas (SP), Brazil R.B.Pinto 521 UEC J.Léonard Guibourtia chodatiana (Hassl.) Corumbá (MS), Brazil R.R.Silva 1153 UEC J.Léonard Guibourtia coleosperma (Benth.) Balovale, Zimbabwe W.Giles 127 WAG J.Léonard *

Guibourtia conjugata (Bolle) Wankie, Zimbabwe R.F.Raymound 218 WAG Downloaded from https://academic.oup.com/botlinnean/article/187/1/87/4965893 by guest on 25 September 2021 J.Léonard * Guibourtia demeusei (Harms) Edea, Democratic Republic of J.Louis 1947 WAG J.Léonard * the Congo Guibourtia hymenaeifolia (Moric.) Oriente, Cuba E.L.Ekman 6203 U J.Léonard Guibourtia hymenaeifolia (Moric.) Cuba Howard & Gonzáles U J.Léonard * 6598 Guibourtia leonensis J.Léonard * Tonkolili, Sierra Leone H.van der Burg et al. WAG 1481 Guibourtia tessmannii (Harms) Jocomba, Equatorial Guinea I.A.Issembé 02 WAG J.Léonard * Hymenaea aff. courbaril L. Óbidos (PA), Brazil R.B.Pinto 471 UEC Hymenaea aff. parvifolia Huber Manaus (AM), Brazil R.B.Pinto 523 UEC Hymenaea courbaril L. Campinas (SP), Brazil V.F.Mansano 175 UEC Hymenaea courbaril L. Manaus (AM), Brazil R.B.Pinto 530 UEC Hymenaea courbaril L. Santana do Tapará (PA), Brazil R.B.Pinto 457 UEC Hymenaea fariana R.D.Ribeiro, Rio de Janeiro (RJ), Brazil (cultivated) A.M. Lino 29 UEC D.B.O.S.Cardoso & H.C.Lima Hymenaea intermedia Ducke Óbidos (PA), Brazil R.B.Pinto 472 UEC Hymenaea intermedia Ducke Manaus (AM), Brazil R.B.Pinto 525 UEC Hymenaea intermedia Ducke Manaus (AM), Brazil R.B.Pinto 533 UEC Hymenaea oblongifolia Huber Barcarena (PA), Brazil R.B.Pinto 498 UEC Hymenaea oblongifolia Huber Rio de Janeiro (RJ), Brazil (cultivated) A.Ducke UEC s.n.(UEC57399) Hymenaea parvifolia Huber Santana do Tapará (PA), Brazil R.B.Pinto 458 UEC Hymenaea parvifolia Huber Oriximiná (PA), Brazil R.B.Pinto 475 UEC Hymenaea reticulata Ducke Manaus (AM), Brazil P.A.C.L.Assunção 537 INPA Hymenaea velutina Ducke Maranhão, Brazil N.Mota 2760 MG Hymenaea verrucosa Gaertn. Rio de Janeiro (RJ), Brazil (cultivated) D.M.Tourinho 23 RB Peltogyne catingae Ducke Manaus (AM), Brazil A.Vicentini 721 INPA Peltogyne discolor Vogel Rio de Janeiro (RJ), Brazil L.F.G.da Silva 85 RB Peltogyne excelsa Ducke Manaus (AM), Brazil M.A.D.Souza 392 INPA Peltogyne mattosiana Rizzini Rio de Janeiro (RJ), Brazil H.C.de Lima 4999 RB

*Fragments of leaflets taken from herbarium sheets. resin (Merck, Darmstadt, Germany). In addition, fresh were performed on fresh material, on herbarium materials from some species were sectioned using razor material and on material embedded in plastic resin. blades for histochemical observation. For herbarium The following chemical reactions were carried out on material, the fragments were hydrated using distilled these materials: Sudan IV (Jensen, 1962) for lipophilic water under vacuum for a couple of days, after which compounds; NADI reagent (David & Carde, 1964) for the procedures for Historesin inclusion were followed. terpenoids; 3% ferric chloride (Johansen, 1940) for Histochemical reactions were chosen based on the phenolic compounds; and ruthenium red (Gregory & chemical classes of higher occurrence in Fabaceae, Baas, 1989) for mucilaginous and pectic substances. especially in representatives of Hymenaea (Crankshaw For analysis with Calcofluor White M2R (Hughes & Langenheim, 1981; Langenheim et al., 1982) and & McCully, 1975) for cellulose, the microscope was

© 2018 The Linnean Society of London, Botanical Journal of the Linnean Society, 2018, 187, 87–98 90 R. B. PINTO ET AL. equipped for epi-illumination with a U-LH100HG (Moric.) J.Léonard (Fig. 1G, H insert) and mercury lamp, providing excitation (bandpass filter G. tessmannii (Harms) J.Léonard (Fig. 1I), the 330–385 nm) and suppression (long-pass filter epidermis accumulates phenolic compounds. Also, 420 nm). The results were documented by capturing in G. chodatiana (Hassl.) J.Léonard lipophilic images from the sections using an Olympus BX 51 substances were observed on the adaxial face of the photomicroscope equipped with an Olympus DP71 epidermis (Fig. 1K insert). The midrib has a vascular camera. cylinder generally composed of one wide vascular bundle and one or more smaller bundles surrounded by a fibrous sheath. In contrast, in the midrib of RESULTS G. hymenaefolia (Fig. 1G) the fibrous sheath is much thicker than in the other species. Between the Downloaded from https://academic.oup.com/botlinnean/article/187/1/87/4965893 by guest on 25 September 2021 The anatomical characterization of the leaflets of 20 epidermis and the vascular bundles, the collenchyma species of the Hymenaea clade is described below and and the mainly regular parenchyma have rounded illustrated in Figures 1–3, according to the genera. to slightly flattened glandular cavities in varied The general characterization of secretory structures frequency according to the species (Fig. 1A, C, E, G, related to the presence of mucilage and the relative I, L, M). In the internerval region, the mesophyll groups of metabolites is listed in Tables 2–4. When is dorsiventral, generally with one to two layers of different individuals from the same taxon were palisade parenchyma and four to six layers of spongy observed, there were no significant variations in parenchyma (Fig. 1B, D, F, H, J, K). Glandular morphology or composition. cavities are more frequent in palisade parenchyma (Fig. 1B, D, F, H insert, J, K insert) but can also occur Guibourtia in the spongy parenchyma (Fig. 1N). In the cavities The leaflets of Guibourtia spp. have an unstratified of Guibourtia, the histochemical reactions indicate epidermis (Fig. 1A–K) frequently composed of the presence of lipophilic substances (Fig. 1K insert, isodiametric cells, wider than they are long, except L), phenolic compounds (Fig. 1M, N) and terpenoids in G. demeusei (Harms) J.Léonard (Fig. 1F), in (Fig. 1O, P). Furthermore, phenolic deposits are which the epidermal cells are longer than they are present in the chlorophyll parenchyma (Fig. 1N) in wide. In G. demeusei (Fig. 1F), G. hymenaeifolia all Guibourtia spp. studied here.

Table 2. Anatomical features related to the presence of mucilage in the leafy tissue of species of the Hymenaea clade

Taxon Mucilaginous idioblasts Accumulated mucilage in the mesophyll Mucilaginous epidermis

Guibourtia chodatiana – – – Guibourtia coleosperma – – – Guibourtia conjugata – – – Guibourtia demeusei – – – Guibourtia hymenaeifolia – – – Guibourtia leonensis – – – Guibourtia tessmannii – – – Hymenaea aff. parvifolia – – – Hymenaea aff. courbaril – + – Hymenaea courbaril – – – Hymenaea fariana – – – Hymenaea intermedia – + – Hymenaea oblongifolia – + – Hymenaea parvifolia – + – Hymenaea reticulata – – – Hymenaea velutina – – – Hymenaea verrucosa – + + (AD) Peltogyne catingae + (ME) + + (AD/AB) Peltogyne discolor + (ME) + + (AD/AB) Peltogyne excelsa – – – Peltogyne mattosiana – – + (AB)

+, Presence; –, absence; ME, all the mesophyll; AB, abaxial face; AD, adaxial face.

Table 3. Chemical classes of substances found in leaves of species of the Hymenaea clade analysed

Taxon Phenolic compounds Terpenoids Lipophilic substances Pectic substances

Guibourtia chodatiana CV, ME, PM CV CV, EP – Guibourtia coleosperma CV, EP, ME, PM CV CV, EP, ME – Guibourtia conjugata CV, ME, PM ? ? – Guibourtia demeusei CV, EP, ME, PM ? CV, EP, ME – Guibourtia hymenaeifolia CV, EP, ME, PM CV CV, EP, ME – Guibourtia leonensis CV, ME, PM ? ? –

Guibourtia tessmannii CV, EP, ME, PM ? ? – Downloaded from https://academic.oup.com/botlinnean/article/187/1/87/4965893 by guest on 25 September 2021 Hymenaea aff. parvifolia CV, EP, ME, PM ? ? – Hymenaea aff. courbaril CV, EP, ME, PM CV, EP, ME CV ME, PM Hymenaea courbaril CV, EP, ME, PM CV, EP, ME CV ? Hymenaea fariana CV, EP, ME, PM ? ? ? Hymenaea intermedia CV, EP, ME, PM ? ? PM Hymenaea oblongifolia CV, EP, ME, PM – – PM Hymenaea parvifolia CV, EP, ME, PM ? ? ME Hymenaea reticulata CV, EP, ME, PM CV CV, ME – Hymenaea velutina CV, EP, ME, PM ? ? ? Hymenaea verrucosa CV, EP, ME, PM – – EP, ME Peltogyne catingae CV, EP, ME, PM ? ? EP, ME Peltogyne discolor CV, EP, ME, PM CV CV, ME EP, ME Peltogyne excelsa CV, EP, ME, PM – CV, ME – Peltogyne mattosiana CV, EP, ME, PM CV CV, ME EP

CV, glandular cavity; ME, mesophyll; EP, epidermis; PM, parenchyma of the midrib; –, absence; ?, data not obtained.

Table 4. Summary and comparative table of sites of secretion of genera of the Hymenaea clade, based in the chemical classes of substances secreted in leaves

Genus Phenolic compounds Terpenoids Lipophilic substances Pectic substances

Guibourtia CV, EP, ME, PM CV CV, EP, ME – Hymenaea CV, EP, ME, PM CV, EP, ME CV, ME ME, PM Peltogyne CV, EP, ME, PM CV CV, ME EP, ME

CV, glandular cavity; EP, epidermis; ME, all the mesophyll; PM, parenchyma of the midrib; –, absence.

Hymenaea and four to five layers of spongy parenchyma, except The leaflets of Hymenaea, in general, have unstratified in H. velutina (Fig. 2I), which has about five layers of secreting epidermis with a predominance of metabolites palisade parenchyma and two or three layers of spongy in all cells, especially phenolic compounds (Fig. 2). parenchyma. The histochemical assays indicated the Epidermal cells are isodiametric, usually wider than presence of phenolic compounds in all the chlorophyll they are long. As in Guibourtia, the midrib, in general, parenchyma, epidermis and epithelium of the cavities, has one wide vascular bundle and one or more small both with toluidine blue (Fig. 2F, 2J) and with ferric bundles surrounded by a sheath of fibres (Fig. 2A, C, E, chloride (Fig. 2M); oleoresin terpenes could be observed G, I, K). In the regular parenchyma and in collenchyma in the cavities, epidermis and idioblasts of the chlorophyll of the midrib, the glandular cavities are located in parenchyma (Fig. 2N). The content of the cavities subepidermal layers facing both surfaces (Fig. 2A, C, exhibits lipophilic substances (Fig. 2O), probably related E, G, I, K), except in H. velutina Ducke (Fig. 2I), which to the oleoresins. Regarding the epidermis, in addition to does not have any cavity. The shapes of the cavities can phenols, H. parvifolia Huber and H. verrucosa Gaertn. vary widely, being cylindrical (Fig. 2B, C) or sometimes (Fig. 2L, P) have a mucilaginous epidermis. Pectic compressed cylindrical (Fig. 2D). The mesophyll in substances are located in the internal periclinal walls the internerval regions is dorsiventral, generally with (Fig. 2P), separated from the cellulosic portion (Fig. 2Q). one layer of palisade parenchyma (Fig. 2D, F, H, L) This mucilage appears to flow from the cells to the

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Figure 1. Anatomical leaf features of Guibourtia spp. in transverse section. Main vein of a leaflet stained with toluidine blue (A, C, E, G, I). Leaflet blade stained with toluidine blue (B, D, F, H, J). Histochemical reactions in the leaflet blade (K–P). A–B, G. coleosperma; C–D, G. conjugata; E–F, G. demeusei; G–H, G. hymenaeifolia; I, G. tessmannii; J, G. leonensis; K–O, G. chodatiana; P, G. hymenaeifolia. Positive histochemical staining: lipophilic substances in the cavities (K–L) and in the epidermis (K insert) indicated by Sudan IV; phenolic compounds in the cavities (M–N) and in the chlorophyll parenchyma (N) stained by ferric chloride; terpenoids in the cavities (O–P) suggested by the NADI reaction. Note to detail of a cavity in the insert to H. Scale bars = A, C, E, G, I: 100 µm; B, D, F, H, J, K–P: 20 µm. cv, cavity; ad, epidermis of adaxial surface; pp, palisade parenchyma; sp, spongy parenchyma. mesophyll, and remains accumulated in the intercellular vascular bundles, one wide and the other narrower. In spaces (Fig. 2L). contrast, P. mattosiana (Fig. 3E) and P. discolor Vogel (Fig. 3G) have a smaller midrib with one large vascular bundle and one to three small bundles. Peltogyne spp. Peltogyne have glandular cavities in subepidermal layers of the As in Hymenaea, the leaflets of all the studied Peltogyne midrib (Fig. 3C, E, G), except P. catingae (Fig. 3A). spp. have a secreting epidermis with a predominance Also, in relation to the composition of the mesophyll, of phenolic compounds (Fig. 3A–H). Epidermal cells P. catingae (Fig. 3B) and P. excelsa (Fig. 3D) resemble are also isodiametric, wider than they are long. In each other with two layers of palisade parenchyma and P. mattosiana Rizzini (Fig. 3F) mucilage was also about five layers of spongy parenchyma. In addition, present in the abaxial surface of the external periclinal P. mattosiana (Fig. 3F) and P. discolor (Fig. 3H) wall of the epidermal cells. Peltogyne catingae Ducke share similar mesophyllic structure with one layer (Fig. 3A) and P. excelsa Ducke (Fig. 3C) have a wide of palisade and four layers of spongy parenchyma. vascular cylinder in the midrib, composed of two Peltogyne catingae (Fig. 3B) and P. discolor (Fig. 3H)

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Figure 2. Anatomical leaf features of Hymenaea spp. Transverse sections of the main vein of a leaflet stained with toluidine blue (A, C, E, G, I, K). Paradermal section of the leaflet blade showing the slightly elongated form of the cavity stained with toluidine blue (B). Leaflet blade stained with toluidine blue (D, F, H, J, L). Histochemical reactions in the leaflet blade (M–O). A–B, H. aff. courbaril; C–D, H. fariana; E–F, H. reticulata; G–H, H. aff. parvifolia; I–J, H. velutina; K–L, M–O, H. courbaril; P–Q, H. verrucosa. Positive histochemical reactions: phenolic compounds in the cavity and in chlorophyll parenchyma identified by ferric chloride (M); terpenoids in the epidermis and in the mesophyll idioblasts indicated by the NADI reaction (N); lipophilic substances in the cavity indicated by Sudan IV (O); pectic substances in epidermis indicated by ruthenium red (P); and cellulose in epidermal cell wall marked by Calcofluor White (Q). Scale bars = A, C, E, G, I: 100 µm; B, D, F, H, J, K–Q: 20 µm. cv, cavity; vb, vascular bundle; ad, epidermis of adaxial surface; ep, epidermis; pp, palisade parenchyma; sp, spongy parenchyma. have large mucilaginous idioblasts dispersed in the DISCUSSION mesophyll. The cavities are isodiametric, i.e. rounded, The morphoanatomical analyses carried out in this in transverse (Figs. 3F, H) and in longitudinal section study show that in the Hymenaea clade there are (Fig. 3I). The species have phenolic compounds some anatomical features found in the leaflets that in all the mesophyll (Fig. 3B, D, F, H), in regular can be used taxonomically and probably be included in parenchyma, collenchyma and epidermis of midrib and phylogenetic considerations. in the epithelium cavity (Fig. 3J). Oleoresin terpenes (Fig. 3K) and general lipophilic substances (Fig. 3L) were observed in the lumen of the cavities. In the mesophyll, the conspicuous mucilaginous idioblasts of Epidermis features P. catingae (Fig. 3B) and P. discolor (Fig. 3H) seem to According to Gregory & Baas (1989) and Potgieter release their stored mucilage, filling the intercellular & Wessels (1998), the presence of mucilage in spaces with pectic substances (Fig. 3M). leaf cells is genetically determined and can be a

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Figure 3. Anatomical leaf features of Peltogyne spp. Transverse sections of the main vein of a leaflet stained with toluidine blue (A, C, E, G). Cross sections of the leaflet blade stained with toluidine blue (B, D, F, H). Paradermal section of the leaflet blade showing the non-elongated form of a cavity stained with toluidine blue (I). Histochemical reactions of the main vein of a leaflet (J–K) and of the blade (L). A–B, P. catingae l; C–D, P. excelsa; E–F, I, P. mattosiana; G–H, P. discolor. Note the mucilage idioblasts in A–B and G–H, as well as the mucilage in the intercellular space in the same samples. Positive histochemical staining: phenolic compounds in the cavity epithelium and in collenchyma and vein parenchyma identified by ferric chloride (J); terpenoids in the cavity indicated by the NADI reaction (K); lipophilic substances in the content of cavity indicated by Sudan IV (L). Scale bars = A, C, E, G, I: 100 µm; B, D, F, H, J, K, L: 20 µm. cv, glandular cavity; co, collenchyma; ad, epidermis of adaxial surface; id, idioblast; pp, palisade parenchyma; sp, spongy parenchyma;. ae, abaxial face of epidermis. useful character in taxonomic studies. Regarding (Castro et al., 2005; Jordan, Dillon & Weston, 2005; the epidermis, all taxa analysed show a secretory Zhang, Zheng & Tyree, 2012). Guibourtia chodatiana function, accumulating phenolic compounds, but only is the only species of the genus present in this kind of H. verrucosa, P. catingae, P. discolor and P. mattosiana habitat (South American chaco and Brazilian cerrado). have mucilage in their epidermis. Representatives Therefore, the presence of lipophilic substances in of H. aff. parvifolia collected in the Reserva Adolpho G. chodatiana may be an adaptation to its habitat. Ducke (Manaus, Brazil) did not have mucilage in their Most Guibourtia spp. occur in moist forests in Africa leaflet epidermis. Despite its taxonomic value, this and Cuba and they do not have lipophilic substances in characteristic does not seem to present a phylogenetic their cells. Considering the predominance of phenolic signal to be interpreted as a synapomorphy and should compounds in the epidermis of the analysed species, be more deeply investigated in the Hymenaea clade. these metabolites have a known protective function, However, when present, the epidermal mucilage mainly against harmful solar radiation, as discussed occurs on the adaxial epidermis in Hymenaea and by several authors (Karabourniotis et al., 1992; the abaxial epidermis in Peltogyne. In Guibourtia, the Kofidis, Bosabalidis & Moustakas, 2003; Lusa et al., only variation in composition of the epidermis among 2014; Silva et al., 2014; Lusa, Da Costa & Appezzato- species is in G. chodatiana, which contains lipophilic da-Glória, 2016b). substances in the cells (absent in the other species analysed) and has a thicker cuticle (Fig. 1K). Lipophilic substances are common in species that occur in dry Idioblasts and mesophyll features and intensely sunny environments (Barros & Soares, Idioblasts with mucilage were detected in P. catingae 2013; Bombo et al., 2016), such as a thick cuticle layer and P. discolor, and an accumulation of mucilage within

© 2018 The Linnean Society of London, Botanical Journal of the Linnean Society, 2018, 187, 87–98 LEAFLET ANATOMY IN THE HYMENAEA CLADE 95 an intercellular space was detected in the mesophyll (1975) had suggested that these resin cavities should of H. parvifolia and H. verrucosa (the mucilage must occur in all the species of the genus and also in have been produced by at least one of the surrounding Guibourtia spp. (Langenheim, 2003). Nevertheless, cells, none of which retains any visible mucilage). of the 20 investigated species of the Hymenaea clade, The taxonomic value of these characteristics is for H. velutina and P. catingae did not show cavities in the now restricted to species delimitation within the leaflet lamina. genera Hymenaea and Peltogyne. The taxonomic As observed in the representatives of the Hymenaea application of the presence of mucilage has been used clade, glandular cavities can be more frequently found in other groups of Fabaceae for generic delimitation just above the adaxial epidermis within the palisade in tribe Crotalarieae (Boatwright et al., 2009) and parenchyma or less frequently within the collenchyma species delimitation in Zornia based on the presence attached to the abaxial epidermis in the midrib. This Downloaded from https://academic.oup.com/botlinnean/article/187/1/87/4965893 by guest on 25 September 2021 or absence of mucilaginous idioblasts and cavities pattern was also observed previously in Peltogyne (Fortuna-Perez et al., 2012). In relation to the presence paniculata Benth. (Ferreira & Flores, 2013). However, of extracellular mucilage, this trait is not commonly it seems to be variable among representatives of described in plants, but it seems to play an important Detarioideae. Ferreira & Flores (2013) observed in role in enabling organs to maintain water potential in Copaifera pubiflora Benth. that the cavities tend to enviroment fluctuations (Morse, 1990; Nobel, Cavelier occur in the centre of the lamina (usually surrounded & Andrade, 1992; Clifford et al., 2002; Chapotin et al., by spongy parenchyma). The same was observed for 2003; Somavilla, Kolb & Rossatto, 2014). C. trapeziifolia Hayne (Milani, Rocha & Teixeira, In addition to characteristics related to the presence 2012) and C. langsdorffii Desf. (Rodrigues, Teixeira of mucilage, the internerval region of the leaflets has & Machado, 2011). The position of the cavities in other features of taxonomic relevance. The taxonomic the lamina of the leaflets seems to have taxonomic value of the number of layers has been shown to be potential; however, it will require more investigation relevant in other legumes for generic delimitation in other genera of the resin-producing clades. No among Myrocarpus, Myroxylon and Myrospermum differences were found related to the histochemical (Sartori & Tozzi, 2002). composition of the glandular cavities among species of Within the genera of the Hymenaea clade, the the Hymenaea clade. observation of both palisade parenchyma and sponger The presence of terpenoids is a common feature parenchyma layers can help in recognizing genera and of resin-producing plants. In Fabaceae, terpenoids also species. For example, in Guibourtia the mesophyll are commonly found in detarioids (Mackinder, 2005). has one or rarely two layers of palisade parenchyma Fougère-Danezan, Maumont & Bruneau (2003, 2007) and four to six layers of spongy parenchyma. suggested that terpene-producing genera occur in In Hymenaea the mesophyll of all analysed species has the Prioria Griseb. clade and Detarieae s.s. In the only one layer of palisade parenchyma and four to five Hymenaea clade, the localization of accumulated layers of spongy parenchyma, except for H. velutina, terpenoids is, to date, the most congruent character a species from the Brazilian caatinga, with about from a phylogenetic perspective (Fig. 4). For instance, five layers of palisade parenchyma and two to three in Peltogyne terpenoids are found only in the cavities, layers of spongy parenchyma. These variations in as in Guibourtia. In the Hymenaea clade, this H. velutina are probably related to the dry, insolated characteristic represents a plesiomorphic character for conditions to which the species is subjected (Levitt, Guibourtia, whereas in Hymenaea terpenoids can be 1980; Marchi et al., 2008). In Peltogyne the mesophyll found not only in cavities but also in the epidermis and of the Amazonian species P. catingae and P. excelsa mesophyll, indicating an anatomical synapomorphy has two layers of palisade parenchyma and five (Table 4). Further studies are required in Fabaceae, layers of spongy parenchyma, whereas P. discolor and especially in the representatives of Detarioideae, to P. mattosiana, from the Atlantic forest, have one layer evaluate the phylogenetic relevance of the localization of palisade parenchyma and four layers of spongy of histochemical products in the leaves. parenchyma.

Histochemical features of FINAL REMARKS the secretory cavities Our results show that some morphoanatomical features Our findings related to the presence and histochemical can be used for species delimitation, especially those composition of the glandular cavities support the related to the presence of mucilage and the region where observations of Langenheim et al. (1982), who it can be found as accumulated in the parenchyma, in reported the presence of ‘resin pockets’ (or ‘cavities’) idioblasts or in the epidermis. The absence of glandular in the leaflets of H. courbaril L. Lee & Langenheim cavities in some species of the Hymenaea clade was not

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Figure 4. Adapted Bayesian majority rule consensus tree from LPWG (2017) showing localization of terpenoids in different tis- sues of the leaflet lamina. The images show cavities in the midrib of representatives of the Hymenaea clade and internerval regions in Hymenaea. Numbers on the branches correspond to posterior probability values. cv, cavity; ep, epidermis; me, mesophyll. expected based on past observations (Lee & Langenheim, REFERENCES 1975; Langenheim, 2003; Rodrigues et al., 2011; Milani Araújo JS, Meira RMSA. 2016. Comparative anatomy et al., 2012; Ferreira & Flores, 2013) and needs to be more of calyx and foliar glands of Banisteriopsis C. B. Rob. deeply investigated in other taxa. Despite the possibility (Malpighiaceae). Acta Botanica Brasilica 30: 112–123. of Guibourtia not being monophyletic (LPWG, 2017), we Arrhenius SP, Langenheim JH. 1983. Inhibitory effects were unable to find anatomical characteristics that are of Hymenaea and Copaifera leaf resins on the leaf fungus, potential synapomorphies of the different lineages of Pestalotia subcuticularis. Biological Systematics and Ecology this paraphyletic genus. However, the findings regarding 11: 361–366. the composition of epidermis cells and the thickness of Barneby RC. 1996. Neotropical Fabales at NY: asides and the fibrous sheath surrounding the vascular bundles oversights. Brittonia 48: 174–187. are characteristics that help to better delimitate the Barros IO, Soares AA. 2013. Adaptações anatômicas em fol- Neotropical Guibourtia spp., contradicting Barneby’s has de marmeleiro e velame da caatinga brasileira. Revista (1996) proposal to recognize only one species in the Ciência Agronômica 44: 192–198. New World. Also, histochemical analyses of the leaves Boatwright JS, Tilney PM, Van Wyk BE. 2009. The generic revealed potential characteristics that can be used to concept of Lebeckia (Crotalarieae, Fabaceae): reinstatement identify genera in the Hymenaea clade and its usefulness of the genus Calobota and the new genus Wiborgiella. South should be investigated for other groups in Detarioideae. African Journal of Botany 75: 546–556. Bombo AB, Appezzato-da-Glória B, Aschenbrenner AK, Spring O. 2016. Capitate glandular trichomes in Aldama discolor (Heliantheae – Asteraceae): morphology, metabo- ACKNOWLEDGEMENTS lite profile and sesquiterpene biosynthesis. Plant Biology (Stuttgart, Germany) 18: 455–462. This work is part of the PhD thesis of R.B.P. and Bruneau A, Mercure M, Lewis GP, Herendeen PS. 2008. was supported by the Conselho Nacional de Phylogenetic patterns and diversification in the caesalpin- Desenvolvimento Científico e Tecnológico (CNPq) ioid legumes. Botany 86: 697–718. through the award of a scholarship, for which he is Caddah MK, Mayer JLS, Bittrich V, Amaral MCE. grateful. The authors also thank Edital REFLORA 2011. Species limits in the Kielmeyera coriacea complex (CNPq proc. 563550/2010-4, FAPESP proc. 2010/52488- (Calophyllaceae) – a multidisciplinary approach. Botanical 9) for financial support. V.F.M. thanks CNPq (process Journal of the Linnean Society 168: 101–115. 312766/2009-2) and FAPERJ (E-26/111.581/2014) for Calvillo-Canadell L, Cevallos-Ferriz SRS, Rico-Arce research support. We are grateful to Dewey Litwiller L. 2010. Miocene Hymenaea flowers preserved in amber for revision of the English text. The authors declare no from Simojovel de Allende, Chiapas, Mexico. Review of conflicts of interest. Palaeobotany and Palynology 160: 126–134.

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