Flora 256 (2019) 92–99 Contents lists available at ScienceDirect Flora journal homepage: www.elsevier.com/locate/flora Are there buds in the roots of Aspidosperma spp. (Apocynaceae)? A comparative morphoanatomical study of underground organs T Eric Yasuo Kataokaa,1, Daniela Martins Alvesa,2, Ingrid Kochb, , Letícia Silva Soutoa ⁎ a Laboratório de Diversidade Vegetal - Departamento de Biologia, Universidade Federal de São Carlos, Rodovia João Leme dos Santos (SP-264), Km 110, CEP 18052-780, Sorocaba, SP, Brazil b Departamento de Botânica, Universidade Estadual de Campinas, Rua Monteiro Lobato, 255, CEP 13083-862, Campinas, SP, Brazil A R T I C LE I N FO A B S T R A C T Edited by Favio Gonzalez Resprouting is an important strategy for plant persistence through time. This strategy is considered a functional trait that allows plants to recover from disturbances such as fire, drought and mechanical injuries. Despite advancements on the ecology of resprouting from underground organs, few studies aimed at elucidating the anatomical nature of these organs, which is key for the proper identification of the nature of these organs. Literature reports and field observations of Aspidosperma spp. with aggregate distribution and individuals physically connected via underground organs indicated that resprouting may occur in species of the genus. Aspidosperma Mart. & Zucc. representatives are trees in the Apocynaceae family, widely distributed in the Neotropics. In this study, we investigated the anatomy of the underground organs of four Aspidosperma spp. aiming at elucidating their anatomical nature. In addition, we monitored for bud formation on the underground organs of the selected species following collections and described the mode of origin and bud development of Aspidosperma cylindrocarpon. Our findings revealed the root nature of bud bearing underground organs of Aspidosperma, which indicates a potential for vegetative propagation via bud-bearing roots in species of the genus, an important trait in the regeneration of plant communities. Keywords: Bud-bearing roots Resprouting rauvofioid grade Aspidospermateae 1. Introduction Resprouting is an important strategy for plant persistence through time, which allows individual plants to survive, and biomes to recover from disturbance regimes (Bond and Midgley, 2001; Pausas and Keeley, 2014). Disturbance such as fire, drought, and mechanical injuries triggers resprouting, a response that involves resourcing of dormant buds and/or de novo development of buds on the surviving plant organs (Clarke et al., 2013; de Moraes et al., 2016; Pausas et al., 2018). Moreover, ecological studies have shown that resprouting rather than regeneration via seedbank represent a competitive advantage in the early stages after disturbance (Clarke and Knox, 2009), thus underscoring the ability to resprout as an important trait in community dynamics. Additional evidence amounts from recent studies that have quantitatively evaluated the significance of resprouting in the regeneration of tropical plant communities (Mostacedo et al., 2009; Ferreira and Vieira, 2017), which showed a significant contribution of resprouters in the composition of the regenerating vegetation. Despite a current focus on understanding the ecology of resprouting from underground organs, relatively few studies aimed at elucidating the nature of the plant organ bearing resprouts (e.g., Bosela and Ewers, 1997; Hayashi et al., 2001; Hayashi and Appezzato-da-Glória, 2009). This is of topical interest considering the vast diversity of specialized organs from which resprouting may occur (Pausas et al., 2018), which underscores the need for refined studies that adequately characterize them. For example, the appropriate characterization and classification of the underground organs are essential for studies addressing questions on the origin and evolution of resprouting structures. A recent study presented a comprehensive review toward a unified terminology based on the nature of these organs (Pausas et al., 2018); they indicated that at least six locations may store buds on the plant: belowground caudexes, fleshy swellings, rhizomes roots, root crown and woody burls. Aspidosperma Mart. & Zucc. is a neotropical genus that includes approximately 74 species in the Apocynaceae family (Castello et al., Corresponding author. E-mail address: ikoch@unicamp.br (I. Koch). 1 Present address: Laboratório de Sistemática Vegetal - Departamento de Botânica, Instituto de Biociências, Universidade de São Paulo, Rua do Matão, 277, CEP 05508-090, São Paulo, SP, Brazil. 2 Present address: Departamento de Botânica, Universidade Estadual de Campinas, Rua Monteiro Lobato, 255, CEP 13083-862, Campinas, SP, Brazil. ⁎ https://doi.org/10.1016/j.flora.2019.05.005 Received 29 October 2018; Received in revised form 2 March 2019; Accepted 13 May 2019 Available online 17 May 2019 0367-2530/ © 2019 Elsevier GmbH. All rights reserved. Flora 256 (2019) 92–99 E.Y. Kataoka, et al. Fig. 1. Morphology of the underground organs in Aspidosperma species. (a) A. australe, underground organ parallel to the soil surface (arrow). (b) A. cylidrocarpon, development of a shoot (arrow). (c) A. camporum with a shoot developed on the underground organ (arrow). (d) A. tomentosum, underground organ connecting two well developed shoots (arrow). reached reproductive maturity, indicated by flowering or fruiting) that were at easily accessible locations (e.g., edge of forest fragments). Collections were carried out in the state of São Paulo, southeast Brazil, in an ecotone zone where Cerrado vegetation and seasonally semi-deciduous forest co-occur (Kortz et al., 2014). The climate is a transition from Cwa (humid subtropical with dry winter and hot summer) to Cwb (humid subtropical with dry winter and hot summer and temperate summer), with average annual temperature of 22 °C (Alvares et al., 2013). Collection procedures were carried out as follow: the area around the trunk base was carefully dug to expose the underground organs located parallel to the soil surface (at variable depths of 15 cm–20 cm). Pieces of these underground organs were collected from up to three individuals of each species. After initial collections, the same individuals were monitored fortnightly or monthly for six months to check for morphological changes in the underground organs, such as swellings or bud formation. If changes did occur, new pieces were collected. At each locality, we collected voucher specimens, which were deposited in the UEC and SORO herbaria (A. cylindrocarpon Kataoka 177 – UEC 192557; A. australe Kataoka 183 – UEC 192558; A. camporum Kataoka 185 – UEC 192559; A. tomentosum Kataoka 409 – SORO 5217). 2019). Species of Aspidosperma are trees that are widely distributed across all vegetation types in the Neotropics, from very humid to seasonally dry forests (Castello et al., 2019), as important components of mature forests (e.g., Aspidosperma polyneuron Müll.Arg.). Reports based on field observations of species of Aspidosperma, namely A. tomentosum Mart. (Ferreira et al., 2015), A. brasiliense A.S.S. Pereira & A.C.D. Castello, and A. polyneuron (Fonseca et al., 2004; Silva et al., 2004), showing aggregated distribution, firstly indicated that resprouting occurs in the genus, with indications that this strategy occurs as a response to disturbance. However, the nature of the resprouting organs in Aspidosperma is unknown. Therefore, we investigated the anatomy of the underground organs of four Aspidosperma spp. with the aim to elucidate their morphoanatomy. In addition, we investigated bud formation on the underground organs of the selected species and described bud development for A. cylindrocarpon. 2. Materials and methods 2.1. Taxon selection, site description and collection Based on preliminary field observations and species occurrence data, we selected four Aspidosperma species: A. australe Müll.Arg., A. cylindrocarpon Müll.Arg., A. camporum Müll.Arg. and A. tomentosum Mart. A. tomentosum is typical from Cerrado vegetation, while the other three species occur in seasonally semi-deciduous forests. Selection of specimens followed the criteria of mature specimens (i.e., plants that 2.2. Anatomical procedures The pieces of underground organs were fixed in FAA 50 (5% formalin, 5% acetic acid, 50% ethanol) (Johansen, 1940) for seven days, 93 Flora 256 (2019) 92–99 E.Y. Kataoka, et al. Fig. 2. Root transverse sections of Aspidosperma australe (a–b), A. cylindrocarpon (c–d), A. camporum (e), and A. tomentosum (f). (a) Central portion of the transverse section; note lack of pith confirming the radicular nature of buds. (b–f) Details of the root stele, showing protoxylem (arrow) externally, and metaxylem internally. mx, metaxylem; px, protoxylem. describe our results simultaneously, highlighting particular characteristics of each species. and stored in a 70% ethanol conserving solution. Samples were then dehydrated in an ethanol series, embedded in methacrylate at approximately 4 °C (Paiva et al., 2011), and transversally and/or longitudinally sectioned with a rotary microtome. Serial sections of variable thickness ranging from 6 to 10 μm were then stained in 0.05% toluidine blue in sodium acetate buffer at pH 4.7 (O’Brien et al., 1964, modified), and mounted in synthetic resin to obtain permanent slides. Histochemical tests were also conducted in fixed and embedded sections. We used lugol (potassium iodide) to detect starch (Johansen, 1940), Sudan IV to identify lipophilic substances (Johansen, 1940), ferric chloride to detect phenolic compounds (Johansen, 1940), and Xylidine Ponceau (pH 2.5) to identify total proteins (Vidal, 1970). The sections were stained for 15 min, and subsequently rinsed and mounted in distilled water for photo documentation. Anatomical sections were examined and photographed with an Olympus BX51 photomicroscope equipped with an Olympus DP71 camera. Photographs of the external morphology of underground organs were taken with a Canon 700D camera, or with a Zeiss Stemi 2000-C stereomicroscope equipped with a Zeiss Axiocam 105 color camera using focus stacking. Anatomical sections were also photographed under polarized light. 3.1. Morphology All analyzed species showed shoots connected via underground organs (Fig. 1a–d). The organs are cylindrical, parallel to the soil surface, and with inconspicuous nodes and internodes (Fig. 1a–d). Aspidosperma cylindrocarpon and A. camporum developed shoots on the underground organs (Fig. 1b–c). Anatomically, all studied organs lack a pith, and the primary xylem maturation was centripetal (Fig. 2a–f). Therefore, the anatomical structure of the resprouting organs is radicular. 3.2. Roots in secondary growth Roots in early stage of secondary growth are delimited by a uniseriate and lignified epidermis, with lignified trichomes (Fig. 3a). Aspidosperma cylindrocapon has a multiseriate epidermis, with up to four cell layers (Fig. 3b). The exodermis occurs adjacent to the epidermis, with secondary walls and larger cells compared to the epidermal ones (Fig. 3a–b). Beneath the exodermis, in the outermost parenchymatic layer, periclinal cell divisions occur in A. cylindrocarpon and A. australe forming the first periderm, and the onset of phellogen, which produces few phellem layers (Fig. 3c). In all studied species, many parenchymatic 3. Results All studied species showed similar morphoanatomy. Therefore, we 94 Flora 256 (2019) 92–99 E.Y. Kataoka, et al. Fig. 3. Transverse sections of Aspidosperma australe (a, c–d, i,), A. cylindrocarpon (b, f, h), A. camporum (e) and A. tomentosum (g) roots. (a–b) Detail of epidermis and exodermis. (c) Initial periderm formation beneath the exodermis. (d–g) Periderm, proliferative pericycle and phloem; note differential sclerenchyma cell arrangement across species (arrows). (h), Proliferative pericycle region showing starch grains stained with potassium iodide; note sclerenchyma cells surrounded by prismatic crystals seen under polarized light. (i) co; cortex; cr, cork; ep, epidermis; ex, exoderm; pc, prismatic crystal; pp, proliferative pericycle; sc, sclerenchyma cell; sg, starch grain; sp, secondary phloem; sx, secondary xylem. latter only differing in terms of higher density of sclerenchyma cells. In A. cylindrocarpon, sclereids and fibers are present as predominantly concentric rings, sometimes isolated (Fig. 3f), while in A. camporum, they are grouped or roughly concentric (Fig. 3g). In the vascular system, the cambium forms a continuous cylindrical sheath, with the secondary xylem being produced inwards and the secondary phloem outwards. The secondary phloem has sieve-tube elements, companion cells and parenchymatic cells of the axial system. Similar to the proliferated pericycle, the secondary phloem contains fibers and sclereids, surrounded by idioblasts with prismatic crystals (Fig. 3d–g). In A. australe, fibers, sclereids and idioblasts containing prismatic crystals occur predominantly isolated (Fig. 3d); in A. cylindrocarpon fibers, sclereids and idioblasts containing prismatic crystals occur as continuous concentric lines (Fig. 3f); in A. camporum they are aggregated (Fig. 3e); and in A. tomentosum fibers, sclereids and idioblasts containing prismatic crystals occur isolated or in lines (Fig. 3g). The secondary xylem has vessel elements, parenchymatic cells and fibers on the axial system (Fig. 2b–f). The parenchymatic rays are uniseriate and heterocellular (Fig. 4d, f). In A. tomentosum we observed tylose on the vessel elements in the inner xylem region. layers are present in the cortex (Fig. 3a–c). As roots develops, a phellogen is formed in the parenchymatic cells of the cortex, while the epidermis, exodermis and cortical cells are compressed and eventually break (Fig. 3c). The phellogen produces many phellem layers centrifugally and one to two layers of phelloderm centripetally (Figs. 3d–g, 4 a, c). The phellem is composed of juxtaposed and rectangular cells, with 15 cell layers in A. australe (Fig. 3d), 17 in A. cylindrocarpon (Fig. 3f), 20 in A. camporum (Fig. 3e), and around 30 in A. tomentosum (Fig. 3g). The protoplast of the phellem cells is hyaline, and cell walls are suberized. In many regions, lenticels were observed (Fig. 4b), with loosely arranged parenchymatic cells. Pericycle cells divide periclinally forming parenchymatic cell layers internal to the periderm. Therefore, a proliferative pericycle remains in the root secondary structure (Fig. 3d–g). The cells of the proliferative pericycle cells are isodiametric, with secondary cell wall, and accumulate starch (Fig. 3h). Sclereids and fibers also occur in the pericycle (3i, Fig. 4e). They have very thick cell walls, and are surrounded by crystalliferous idioblasts (Fig. 3i). In transverse section, sclerenchymatic cells are differentially organized across species. A. australe (Fig. 3d) has isolated or sparsely grouped sclerenchymatic cells adjacent to the secondary phloem, similar to A. camporum (Fig. 3e), the 95 Flora 256 (2019) 92–99 E.Y. Kataoka, et al. Fig. 4. Transverse (a–b) and longitudinal (c–f) sections of Aspiosperma australe (f), A. cylindrocarpon (d–e), A. camporum (a–b) and A. tomentosum. (c) Periderm; note various phellem layers, phellogen (★) and a lenticel (a, c -b). (d) Detail of the secondary vascular structure; note the xylem ray. (e) Proliferative pericycle region showing fibers and sclereids. (f) Xylem in tangential section, with uni- and biseriate rays (arrows). cr, cork; fi, fiber; ph, phelloderm; sl, sclereid. cells (Fig. 5g) and in the parenchymatic cells of the cortex. We also observed a differential proliferation of the cambium cells (Fig. 5f, h–i), from which vascular elements differentiated independently both acropetally and basipetally (Fig. 5d–g). The vascular elements can eventually connect the bud to the stele of the main root. 3.3. Mode of origin of root buds in Aspidosperma cylindrocarpon During the six-month period, we observed bud and shoot formation in Aspidosperma cylindrocarpon and calli formation in A. tomentosum. In the latter, well developed shoots were found in the field, but no bud or shoot formed during the time period of our study. Therefore, we describe root buds that formed in A. cylindrocarpon. These buds formed in regions where structural damage was inflicted by collection of roots during sampling (i.e., localized sectioning with pruning shears), or when specimens occurred in areas with signals of disturbance (i.e., near dirt roads with occasional tractor traffic, areas subjected to livestock trampling). Along the exposed roots, bud formation sites were predominantly close to wound location. These sites were swollen (Fig. 5a), followed by fissures (Fig. 5a–b) and a bud with leaf primordia (Fig. 5b). The changes detected on the external morphology of bud-bearing roots were anatomically examined in order to observe the origin of root buds in A. cylindrocarpon (Fig. 5c–i). During development, buds grow outwards, tearing the sclerenchymatic rings and the periderm (Fig. 5c–d). Bud formation initiates from a callus that differentiates from the parenchymatic cells of the proliferative pericycle (Fig. 5c), reaching the stage of leaf primordia differentiation (Fig. 5d–e). In addition, we observed anticlinal divisions in the parenchymatic phloem 4. Discussion Our anatomical analyses revealed that the examined organs have centripetal maturation of the primary xylem and lack pith, both indicative of a root structure (Peterson, 1975; Hayashi et al., 2001). The main anatomical differences found in the roots across Aspidosperma species are (1) the number of protoxylem poles, which varied among and within species (three to twelve, depending on the specimen analyzed); (2) the amount of idioblasts containing prismatic crystals of calcium oxalate (which was higher in A. tomentosum); (3) the occurrence of a seasonal cambial activity in A. tomentosum; (4) the presence of two periderms in A. cylindrocarpon and A. australe, three in A. tomentosum during secondary growth establishment and (5) a low accumulation of starch in the parenchymatic cells in A. tomentosum. For Aspidosperma species, several reports of bud formation in both underground and aboveground organs can be found in the literature 96 Flora 256 (2019) 92–99 E.Y. Kataoka, et al. Fig. 5. Morphoanatomy of Aspidosperma cylindrocarpon bud-bearing roots, showing the mode of origin of root buds. (a) Lateral view of an emerging callus with periderm rupture. (b) Bud emergence showing leaf primordia and organ intumescence. (c) Callus, longitudinal section showing proliferative pericycle with acropetal differentiation of the vascular elements (arrow). (d) Bud, longitudinal section showing periderm rupture, leaf primordia differentiation, proliferative pericycle and vascular elements in the median region. (e) Root bud, longitudinal section showing basipetal and acropetal differentiation of vascular elements along the bud longitudinal axis. (f) Detail of vascular element differentiation connecting the bud to the stele of its corresponding root. (g) Detail of anticlinal divisions of secondary phloem parenchymatic cells (arrowhead), vascular element differentiation (arrow) and sclerenchymatic ring rupture. (h–i) Detail of cambial proliferation. lp, leaf primordia; pd, periderm; pp, proliferative pericycle; sp, secondary phloem; sr, sclerenchyma ring; sx, secondary xylem; ve, vessel element; vc, vascular cambium. 97 Flora 256 (2019) 92–99 E.Y. Kataoka, et al. the publication of this paper. (Vieira et al., 2006; Medeiros and Miranda, 2008; Mostacedo et al., 2009). These studies reported the development of buds and/or shoots, both on stems and underground organs in A. cylindrocarpon, A. pyrifolium Mart., A. quebracho-blanco Schltdl., A. rigidum Rusby and A. subincanum Mart. In our study, we provide the first anatomical description of bud-bearing underground organs in the genus. We identified the root structure of the organs, which in conjunction with other characteristics such as lateral, cylindrical roots, parallel to the soil surface, without conspicuous nodes and internodes, and with vertical suckers, are traits consistent with bud-bearing roots (Rizzini and Heringer, 1966; Pausas et al., 2018). Bud-bearing roots were also identified in tree species from Cerrado vegetation and seasonally semideciduous forests (Hayashi et al., 2001; Hayashi and Appezzato-daGlória, 2009; Imatomi et al., 2014). Among the studied species, only Aspidosperma cylindrocarpon developed buds on roots during the six-month period we monitored the individuals. Following the terminology proposed by Bosela and Ewers (1997), we characterized the root buds in A. cylindrocarpon as reparative, as they are formed de novo in roots in secondary growth, in response to injuries. Sites of root bud initiation are quite variable among species, with various tissues being involved in the development of bud primordia (Peterson, 1975). In A. cylindrocarpon, buds are formed through the differential proliferation of the cambium and the pericycle, with no vascular connection to the stele of the parent root; thus, these buds are considered exogenous in origin (Bosela and Ewers, 1997; Hayashi et al., 2001). The absence of buds on roots of the remaining three Aspidosperma species does not necessarily indicate that they do not produce root buds. We suggest that our monitoring period was not sufficient to detect the development of root buds in A. australe, A. camporum and A. tomentosum, especially considering seasonality of temperature and precipitation in the study area. The monitoring period (April to September) corresponded to months of low temperature and low average precipitation (Alvares et al., 2013). For these species, instead of buds, we verified mature individuals and resprouts physically connected in the field. In addition, root sprouts in A. tomentosum were found in an area subjected to controlled burning (Medeiros and Miranda, 2008), and resprouting from root cuttings was verified for A. camporum in a restoration project (Ferreira and Vieira, 2017), thus suggesting that budbearing roots also occur in the remaining three examined species. Resprouting is an important strategy for plant persistence through time, being recognized as a functional trait (Clarke et al., 2013). This strategy is particularly advantageous in ecosystems subjected to frequent fire, which is a severe, biomass depleting disturbance (Clarke et al., 2013; Bond and Midgley, 2001; Ferreira et al., 2015; Kammesheidt, 1999). In disturbance regimes, resprouting may be an advantage compared to seedling recruitment because resprouts are not necessarily affected by the same pressures to which seedlings are subjected to in their establishment phase, such as desiccation and predation (Vieira et al., 2006). Therefore, species with the ability to resprout may contribute to vegetation regeneration after disturbance (Mostacedo et al., 2009; Ferreira and Vieira, 2017). Funding The research was supported by FAPESP with a scholarship granted to E.Y.K. (2014/19175-8) and by CNPq with a scholarship granted to D.M.A. (118833/2014-6). Acknowledgments The authors thank F.F. Mazine and A.H. Hayashi for critically reviewing an earlier version of the manuscript; and to A.C.D. Castello, J.D. Vidal, and F. Gueiros for field work assistance. References Alvares, C.A., Stape, J.L., Sentelhas, P.C., Gonçalves, J.L.M., Sparovek, G., 2013. Koppen’s climate classification map for Brazil. Meteorol. 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