The Amazonian Croton mollis (Euphorbiaceae): morphology and leaf anatomy help to understand its preference for the extreme igapó habitat

Flora 281 (2021) 151878 Available online 27 June 2021 0367-2530/© 2021 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). The Amazonian Croton mollis (Euphorbiaceae): morphology and leaf anatomy help to understand its preference for the extreme igap´ o habitat Narah Costa Vitarelli a , Nadia Somavilla b , Fl´ avia Bonizol Ferrari b , Matheus Rezende e Silva b , c , Ester Moreira Soares a , d , Ot´ avio Luis Marques da Silva e , Ricarda Riina f , * a Departamento de Educaç˜ ao e Ciˆ encias, Núcleo de Biologia, Instituto Federal de Ciˆ encias e Tecnologia do Sudeste de Minas Gerais (IF Sudeste MG, Campus Juiz de Fora), Rua Bernardo Mascarenhas, 1283, Bairro F´ abrica, Juiz de Fora, Brazil b Departamento de Botˆ anica, Universidade Federal de Juiz de Fora (UFJF), Brazil c IC Scholarship in PROPP/UFJF, Brazil d BICJr. Scholarship in PROPESQUINOV FAPEMIG from IFSUDESTMG, Campus Juiz de Fora, Brazil e Instituto de Botˆ anica, Núcleo de Pesquisa Curadoria do Herb´ ario SP, Avenida Miguel St´ efano, 3687, S˜ ao Paulo, Brazil f Real Jardín Bot´ anico, RJB-CSIC, Plaza de Murillo 2, ES-28014, Madrid, Spain ARTICLE INFO Edited by: Dr. Favio Gonzalez Keywords: Anatomy environmental adaptation Euphorbiaceae secretory structures systematics trichome-like emergences ABSTRACT Identifying traits contributing to the success of organisms living in harsh environments has always been of great interest in evolutionary ecology. Here we focused on morphological features that could be advantageous for the survival of Croton mollis, a slender shrub occurring in the extreme white-sand habitats of the Amazonian igapos (black/clear-water rivers). We examined macro-morphological characters, as well as leaf micro-morphology using light microscopy and scanning electron microscopy. We also estimated areas of habitat suitability for C. mollis based on climatic, soil, and elevation variables using species distribution modelling (SDM) and available herbarium collections. Our results show that C. mollis presents morphological attributes similar to those of rheophytes, which may allow the species to overcome drastic seasonal changes in water level. We report the presence of five secretory structures (extrafloral nectaries, colleters, idioblasts, glandular trichomes, and laticifers) in the leaves. The size and position of the secretory idioblasts support the hypothesis of the existence of a transitional storage for secondary metabolites from idioblasts to secretory trichomes. Besides glandular trichomes, we also found stellate trichomes and trichome-like emergences in leaves. We hypothesize that these emergences could have an important role in the species’ survival due to their capacity of absorbing atmospheric moisture. Finally, the SDM supported the known habitat preferences of C. mollis and estimated a relatively wider geographic range than the currently known distribution based on herbarium records, suggesting that collecting efforts in the Amazonian region need to be increased in the future. 1. Introduction Amazonian white-sand ecosystems constitute an example of a naturally fragmented habitat with extreme soil conditions (i.e., substrates extremely poor in nutrients, with high permeability and low water retention) and represent a unique component of Amazonian biodiversity (Anderson, 1981; Adeney et al., 2016). It is recognized that this kind of inland island-like habitats could have favored speciation (Adeney et al., 2016) and that plants growing on them can serve as model systems to understand the role of edaphic adaptation in ecological speciation (Rajakaruna, 2018). Studies on the evolution of plant groups in Amazonian white-sand ecosystems are scarce, being Pagamea Aubl. (Rubiaceae) (Vicentini, 2016) and Protium Burm.F. (Burseraceae) (Fine et al., 2014) some of the best studied genera. Both genera are important elements of the species-poor but highly endemic flora associated with these oligotrophic habitats (Fine et al. 2010). Although less studied, the genus Croton L. (Euphorbiaceae) is another interesting case, with numerous species endemic to these white-sand areas (e.g., Secco, 2004; Secco and Berry, 2010; Secco et al., 2012; Sodr´ e and Silva, 2018; Sodr´ e et al., 2019a; Riina et al., 2021). Among the Amazonian white-sand ecosystems, the Igap´ o refers to seasonally floodplain forests and open vegetation occurring in acidic, nutrient-poor soils along black (e.g, Atabapo, Rio Negro) or clear (e.g., Rio Branco) water rivers in the Amazonian region (Prance, 1979, 1980; * Correspondence author. E-mail address: [email protected] (R. Riina). Contents lists available at ScienceDirect Flora journal homepage: www.elsevier.com/locate/flora https://doi.org/10.1016/j.flora.2021.151878 Received 14 January 2021; Received in revised form 22 June 2021; Accepted 22 June 2021 Flora 281 (2021) 151878 2 Oliveira-Filho et al., 2021), which in most cases are the result of fluvial deposits of different ages from the Guiana and Brazilian shields (Adene et al., 2016; Oliveira-Filho et al., 2021). In general, the igap´ o forest vegetation is well preserved since the local economy around them is mainly restricted to the extraction of non-timber forest resources and fishery (Emperarie, 2000; German, 2004). However, igap´ os close to urban areas such as Manaus, are threatened by anthropogenic actions such as urban sprawl, sport fishery, river beaches, and ecotourism (Montero and Latrubesse, 2013). The most common Croton species occurring in open or forest vegetation associated with igap´ os are Croton cuneatus Klotzsch (tree), C. mollis Benth. (shrub), C. sacaquinha Croizat (shrub, treelet), and C. yavitensis (tree) (Riina et al., 2010; Caruzo et al., 2020). Some of these, particularly the trees, are not exclusive of igap´ os, but also occur along white-water Amazonian rivers. Here we focus on C. mollis, a species restricted to open vegetation on sandy and rocky sites along igap´ os, in northern Brazil and southern Venezuela-Colombia. In the studied site, C. mollis appears as a locally dominant species along the Rio Negro near Manaus (Amazonas, Brazil) where individuals grow in ecologically challenging conditions, including extremely poor, sandy substrates, with high permeability and low water retention (Anderson, 1981; Oliveira-Filho et al., 2021), together with high flood level variation. Plants limited to streambeds within the flood level are known as rheophytes. They can occur under torrential conditions in rapids, cascades, on gravel and sand bars, and even in relatively slow streams (van Steenis, 1932, 1978). Rheophytes have evolved in many angiosperm families, but appear to be more common in Euphorbiaceae, Rubiaceae, and Myrtaceae (van Steenis, 1978). Although Euphorbiaceae is rich in rheophytes (e.g., Berry and Wiedenhoeft, 2004; Cheek et al., 2016), very few Croton species have been documented as part of this ecological group. The Old World “Riau Pocket” Croton clade with six species distributed in Borneo and Thailand (Airy Shaw 1972; Esser, 2010; van Ee et al., 2015), is one example of a rheophytic Croton lineage. Until recently, C. mollis was a poorly known shrub with the only morphological information coming from brief or succinct descriptions from the protologue (Bentham, 1854) and the Flora of the Venezuelan Guayana (Berry, 1999). New taxonomic and geographic information about this species has been recently published (Sodr´ e et al. 2019a; Riina et al., 2021), however the species morphological description was not updated in those studies because of their synoptical approach. Our main aim with this study was to understand how Croton mollis copes with the environmental constraints of the igap´ o habitat by identifying putative adaptations or functional traits at the macro and micromorphological scales. We also provide new information that completes the species’ botanical description, update its geographic range by verifying available herbarium collections, and estimate habitat suitability using species distribution modelling (SDM) to confirm its restriction to Amazonian igap´ os. 2. Materials and methods 2.1. Study site and sampling We studied a population of Croton mollis from the banks of the Rio Negro in the Municipality of Manaus (Amazonas, Brazil), near the Ponta Negra beach (approximate coordinates: 3◦03′33.6"S 60◦06′41.0"W). This population is characterized by slender, few-branched shrubs of 1-2 m tall growing on a rock outcrop with a thin layer of sandy soil originated from the Rio Negro sediments (Fig. 1). During the rainy season (November to February), the water level rises and this site becomes Fig. 1. The studied population of Croton mollis on the banks of the Rio Negro in a locality near the city of Manaus. A, B. Shrubs of 1-2 m tall with etiolated branches, growing on a rock outcrop with a thin layer of sandy soil. Individuals of Croton mollis dominate the local landscape. Note the presence of etiolated branches and nodes and leaves concentrated at the branch tips. C. Branch with inflorescences, showing pistillate and staminate flowers, and leaves. D. Stenophyllous leaves with nectaries at the leaf base (arrows). N.C. Vitarelli et al. Flora 281 (2021) 151878 3 completely flooded. Mean monthly temperatures vary little over the year and range between 25◦C and 28◦C (Sombroek, 2001). In the Manaus area, the water level of Rio Negro oscillates 15-16 m yearly between the highest and lowest levels (Montero and Latrubesse, 2013), which means that the C. mollis population could be partially or totally flooded during the highest peak of the rainy season. Croton mollis was collected in October 2013. Voucher specimens (Vitarelli 013, 014, 015, 016, 017, 018) were deposited in the IF Sudeste MG plant collection and the CESJ herbarium (Table A1). Besides the collected material used for the anatomical studies, we also surveyed all the available herbarium collections of C. mollis (A, B, BM, BR, CESJ, COAH, EAFM, ESA, HBG, G, GOET, INPA, L, LD, LE, MIRR, MO, NY, OXF, P, PORT, RB, TCD, UEC, UNIP, US, VEN) (acronyms follow Thiers, 2021, continuously updated), including unidentified and misidentified specimens, to analyze morphological features and update the species distribution range. All the specimens were examined and verified against the type material and the species original description (See supplementary material, Table A1). Specimens were georeferenced using locality data and original coordinates included in herbarium labels (converted to decimal degrees when necessary through conversor [splink.cria.org.br/conversor]). Specimens lacking coordinates or with doubtful ones were georeferenced using the geoLoc tool of speciesLink (http://splink.cria.org.br/geoloc) and searches on Google Maps or georeferenced collections from the same locality. Species Distribution Modeling (SDM) was performed on MaxEnt v. 3.4.1. (Phillips et al., 2017), with auto features and logistic output. Performance of the projections were evaluated by area under the curve (AUC) on receiver operating characteristics (ROC) and the variables used for projecting habitat suitability for C. mollis included climatic with high resolution (30 arc-sec) from CHELSA (Karger et al., 2017a, b), soil qualities from Food and Agriculture Organization of the United Nations (Fischer et al. 2008) and elevation from NASA Shuttle Radar Topographic Mission (SRTM). The distribution map, including the results from the SDM, was created with the software QGIS 3.4.3 (Quantum GIS Development Team, 2019). 2.2. Foliar Index Foliar index (FI) is a measure used to evaluate the stenophyllous character of leaves. This index is commonly used in studies of rheophytes and was proposed by van Steenis (1978). Stenophyllous leaves are long and narrow with a FI (length:width ratio) ≥3 (van Steenis, 1978). The FI was calculated using the following equation: FI =L/W, where L is the length and W is the width of the leaf blade (van Steenis 1978). Fifteen herbarium specimens of C. mollis (Table A1), including three of the collected by the first author (Vitarelli 013, 014, 015), were used to obtain the measures to estimate the foliar index. We measured two fully developed leaves per specimen (n =30 leaves). 2.3. Light microscopy Samples were obtained from the herborized material described in section 2.1 (Vitarelli 013, 014, 015, 016, 017, 018) to perform a morphoanatomical characterization of leaves. It is important to mention that after field collecting, these herbarium specimens were air-dried, i.e., we did not use a fixed solution (for anatomical studies) nor an ethanol solution (traditionally used when collecting in remote tropical regions). For the description of surface characters, whole leaves from the dried herbarium material were hydrated (Smith and Smith, 1942), and then cleared with 10% sodium hydroxide, followed by 20% sodium hypochlorite (Johansen, 1940, modified). The tissues were stained with safranin (1% alcoholic solution) and mounted in glycerinated gelatin. The classification and terminology of trichomes followed Webster et al. (1996) and Vitarelli et al. (2016). Transverse sections of the middle portion of leaves were submitted to hydration solution for herbarium samples (distilled water at room temperature; ethanol; glycerine; detergent; 100:80:20:0,25 v/v) for 48 h, keeping samples in stove (60◦C) for 2 hours, and then washed in distilled water (ULM, Germany, J.E.A. Mariath, personal information). The sections were obtained with a hand microtome (Ranvier model), cleared with sodium hypochlorite 20%, washed in distilled water, stained in aqueous astra blue (1%) followed by alcoholic safranin (1%), dehydrated in ethanol series (50, 70, 90, 100%), followed by ethanol/ xylene solution (3:1, 1:1, 1:3) and xylene, and finally mounted in colorless varnish (Paiva et al. 2006). Part of the hydrated samples were embedded in paraffin (Johansen 1940), sectioned (10-12 µm) with an Ancap model 297 rotary microtome, deparaffinized and stained with safranin and astra blue, followed by dehydration through an ethanol/xylene series (modified from Gerlach, 1984), and mounted using a colorless varnish medium (Paiva et al., 2006). Histochemical tests were used to confirm the chemical nature of the cell wall structures. Entire leaves were rehydrated (Smith and Smith, 1942), then hand-cut sections were obtained from samples for performing the following histochemical tests: Sudan IV (Pearse, 1980) for detection of total lipids; phloroglucinol for lignin (Johansen, 1940); and Rutenium red (Johansen, 1940) for pectins. Observations, image capture, and photographs were performed on an optic microscope (Zeiss AX10 model coupled with an AxioCamERc 5S camera). 2.4. Scanning electron microscopy To allow detailed observations of external leaf structures, four herbarium samples (Vitarelli 013, 014, 015, 016) were fixed in stubs using carbon tape, sputter coated with gold (Leica EM SCD050) and analyzed using scanning electron microscopy. We used the Tescan, Vega 3 SBU Easy Probe electronic microscope from the Núcleo de Metalurgia of IF Sudeste MG (Campus Juiz de Fora) and the JEOL JSM 7000F electronic microscope from the Microscopy and Microanalysis Laboratory of IB/ Universidade de Brasília (UnB). 3. Results 3.1. Species distribution, habitat, habit, and foliar index The map of the verified herbarium specimens of Croton mollis resulted in two areas of concentration of collection points, one located within the Rio Negro basin in Brazil (along Rio Negro and black-water tributaries of the Branco river), and the other in the basin of the Atabapo river along the border between southern Venezuela and Colombia (Fig. 2). The list of herbarium specimens with approximate coordinates is included as supplementary material (Table A1). According to the information from specimens, the habitat of C. mollis is mostly associated with black rivers, and collectors include the following habitat descriptors in herbarium labels: river banks, flooded areas, sandy substrates, river beaches, rock outcrops, and hydromorphic soils (Table A1, Figs. 1 and 2). The species distribution modeling (SDM) projection showed good overall performance (AUC =0.993) and revealed potential areas of occurrence for C. mollis (still unrecorded in herbaria), with high climatic suitability, including the vicinities of Bel´ em (Par´ a, Brazil), and around the mouth of rivers between Tapaj´ os and Madeira Rivers along the Amazon River (Fig. 2). Among the variables used in the SDM, the ones that contributed the most to the model (Table 1) were precipitation of the coldest quarter (29.3%), annual mean temperature (21.5%), altitude (11.6%), precipitation of the driest month (11.1%), and soil oxygen available to roots (6%). Based on the examination of herbarium specimens and field observations, we provide new morphological data for Croton mollis. The species is a 1-2 m shrub with etiolated branches. Leaves are (2.3)3-4.2 (6) cm long, (0.6)0.8-1.3(1.7) cm wide, discolorous, elliptical to lanceolate with entire margin (matching those of the lectotype Spruce 1806 and other collections such as Prance 19416 and Rodriguez 2043), some individuals have ovate to obovate leaves with dentate margin (e.g., N.C. Vitarelli et al. Flora 281 (2021) 151878 4 Forzza 6493, Vicentini 1461). Leaves are generally clustered at the most distal nodes (Fig. 1B). The foliar index (FI: mean =3.8, median =3.4, standard deviation =1.2, n =30) is consistent with the stenophyllous leaf type (FI ≥3). Extrafloral nectaries are present both at the apex of the petiole and along the leaf margin (see anatomy results). The tiny nectaries along the leaf margin fall off easily during plant pressing and are commonly missing in herbarium specimens; for example, only one marginal nectary was detected in one of the Spruce 1806 sheets at K (M. Luj´ an, pers. comm.). For this reason, the presence of marginal nectaries cannot be used alone to identify C. mollis. Young branches and leaves are covered by a dense indumentum of stellate trichomes (see details in the anatomy section). Inflorescences are short, 3-4(7) cm long, with one or a few pistillate flowers at the base of the inflorescence axis, and staminate flowers on the upper portion; in most cases pistillate and staminate flowers are separated by a naked portion (lacking flowers) of the inflorescence axis. Information on the taxonomy of C. mollis, including new synonyms, is available in a recent taxonomic synopsis of the entire C. section Geiseleria (Riina et al., 2021). 3.2. Leaf anatomy Leaves are amphistomatic and present a unicellular epidermis. The abaxial leaf surface epidermis presents secretory and stellate trichomes (Fig. 3), whereas on the adaxial side, there are only stellate trichomelike emergences (Figs. 3 and 4). Trichomes and emergences are further described below. The mesophyll is dorsiventral with one layer of palisade parenchyma and 5 to 7 layers of spongy parenchyma (Fig. 3F, 3G). Secretory idioblasts (Figs. 3G and 6) and many idioblasts with druses are spread in the mesophyll (Fig. 3F). 3.3. Micromorphology of the indumentum Leaves are covered by indumentum on both surfaces (Fig. 3). The abaxial surface is covered by stellate trichomes (Figs. 3B, 3D, 3E, 3F, 3H), which present a stalk and have lateral rays organized in two levels, each level with less than 8 rays (Figs. 3B, 3D, 3H). The rays have a thick, secondary, lamellate and usually lignified cell wall, as shown by the floroglucin test results; however, some rays do not show lignified ends. At the base of each ray, the cell wall is pitted allowing strong communication between the cells of the base of trichomes. The trichome stalks are variable in length. The shorter stalks usually have cell walls completely lignified (Fig. 3H) and the longer ones have the apical region with cell walls lignified whereas the basal part has cell walls with lipidic compounds, as shown by the positive reaction to floroglucin and sudan histochemical tests, respectively. Fig. 2. Map showing the approximate locations (dots) of the known herbarium records of Croton mollis in South America (data source in Table A1) and habitat suitability for this species based on SDM using climatic, soil and elevation variables. Table 1 Estimates of relative contributions of the environmental variables to the SDM Maxent model for Croton mollis. Variable Contribution (%) Precipitation of coldest quarter 29.3 Annual mean temperature 21.5 Altitude 11.6 Precipitation of the driest month 11.1 Oxygen availability to roots 6 Mean temperature of the coldest quarter 5.6 Precipitation of warmest quarter 3.8 Precipitation of the wettest month 3.6 Nutrient retention capacity 3 Precipitation of the driest quarter 1.9 Temperature seasonality 1.2 Temperature annual range 0.4 Workability (constraining field management) 0.3 Precipitation seasonality 0.3 Max temperature of the warmest month 0.1 N.C. Vitarelli et al. Flora 281 (2021) 151878 5 The adaxial leaf surface is covered by trichome-like stellate emergences (Figs. 3A, 3C, 3E-G and 4). The term “emergence” is used because of the presence of an outer and an inner region (Fig. 4A). The outer region is formed by a central ray (porrect ray) and lateral rays, linked to a sclereid in the inner region (mesophyll). These emergences are sessile, although they present a set of cells at the base forming a cushion (Figs. 3C, 3E-G and 4A, 4C, 4D). The cuticle covers the ordinary epidermal cells and cushion cells, but the results of the Sudan reaction under light microscopy indicate that the cuticle on the ray cells is absent or so thin that it could not be detected (Fig. 4D). The outer region is characterized by a single large central porrect ray, and short lateral rays which are variable in number (Figs. 3A, 3C, 3E-G and 4A, 4C and 4D). The porrect ray is variable in length, approximately five times the length of lateral rays (Fig. 3F). The rays cells appear to be dead and present thick secondary pitted walls with strongly lamellar structure (Fig. 4B). The ray cell wall remains non-lignified in their main extension, showing lignin deposition in the basal region of the ray (Fig. 3F), which is intensely pitted (Fig. 4D). That region is connected with sclereids from mesophyll (Fig. 4A, 4C), which also present thick, secondary, pitted and lignified cell walls (Fig. 4C). Unlike the ray cells, fully formed sclereids seem to remain alive. Strong evidence supporting this is the presence of cytoplasmic content in some of these cells (Fig. 4C), the conspicuous nuclei (Fig. 4C), and the numerous structures that could be plasmodesmata connecting sclereid cells with adjacent cells of the mesophyll (Fig. 4E). Nevertheless, both cytoplasmic metabolism and communication between plasmatic membranes should be confirmed using electron transmission microscopy. Sclereids can reach vascular bundles cells or sheath bundle cells (Fig. 4E) or else pass through the entire mesophyll (Fig. 3G). 3.4. Secretory structures Below we describe the five different leaf secretory structures identified in C. mollis. Extrafloral Nectaries (EFNs) – We observed EFNs at the base of leaves, in two or more pairs (Fig. 1D and Figs. 5A-F) and along the leaf margin (Fig. 5G). The EFNs are stalked and their secretory portion is located at the distal region, which presents a concave surface (Figs. 5AG). Extrafloral nectaries are intensely vascularized by bundles from the petiolar vascular system, which reach the distal region at a subepidermal position. Laticifers to accompany the vasculature of EFNs (Fig. 5F), and idioblasts containing druse-type crystals are present in the stalk (Fig. 5E, 5G). Colleters – These structures are persistent in the mature leaves and Fig. 3. Leaf indumentum in Croton mollis. A - E. Scanning electron microscopy. A, C. Stellate emergences on adaxial surface. B, D. Stellate trichomes on abaxial surface; note the lateral rays organized in two levels. E. Transverse section showing both stellate emergences and stellate trichomes. F - H. Light microscopy of leaf section. F. Transverse section showing emergences and trichomes distribution. G. Unicellular epidermis and dorsiventral mesophyll; note stellate emergence with sclereid traversing the mesophyll and a conspicuous idioblast interspersed with palisade and spongy parenchyma; see also the secretory trichome on the abaxial surface. H. Detail of stellate trichome in abaxial surface. Abbreviations: Yellow arrowheads point to ray cells from the upper level; blue arrowheads indicate ray cells from bottom level; Aba, abaxial leaf surface; Ada, adaxial leaf surface; Id, secretory idioblast; SE, stellate emergence; SeT, secretory trichome; ST, stellate trichome; ], delimitation of the one-layered epidermis. N.C. Vitarelli et al. Flora 281 (2021) 151878 6 occur along the leaf margins (Figs. 5E and 5H-J). Their structure follows the standard type, composed of a short stalk, a parenchymatous axis (with many cells containing druses), and recovered by a palisade secretory epidermis. Laticifers – Although we did not observe latex secretion in the field, laticifers in the leaves of this species occur associated with the vascular system. They were observed in nectaries (Fig. 5F) and stem vasculature (Figs. 5K). Laticifer cells were elongated and organized in rows constituting articulated laticifers. It was not possible to define whether laticifers were anastomosed or not in C. mollis. Secretory idioblasts – These are present in high densities in leaves, and differ in shape/size, position, and distribution (Fig. 6). There were conspicuous idioblasts in the mesophyll (Figs. 6C-F), some interspersed with the palisade cells oriented towards the adaxial leaf surface with part of the cell in contact with the external surface of the leaf (Figs. 6D, 6E, both indicated by arrowhead), others occurred only between spongy parenchyma cells, oriented towards the abaxial leaf surface (Figs. 6F, 6G), or positioned across the mesophyll, being part of both palisade and spongy parenchyma, and covering a large part of the mesophyll (Fig. 6E). Other idioblasts seem to occupy a transitional position between the mesophyll and the epidermis. In some cases, we could not be certain if the observed structure was a mesophyll idioblast or an Fig. 4. Light microscopy of leaf transverse sections showing structure of an stellate emergence in Croton mollis. A. General view of the stellate emergence showing the outer and inner regions. B. Detail of ray thick secondary cell wall, showing its strong lamellar structure. C, D. Detail of joining outer and inner regions; note connections between rays (outer region) and sclereids from the mesophyll (inner region), which are densely pitted. C. Ray cells with thick, lamelate and pitted walls; note sclereids with thick and pitted cell walls, dense content in the cytoplasm of sclereid cells, and conspicuous nuclei. D. Lamelations on the ray cells walls and the apparently strong communication between ray cells and sclereids through pits in the cell wall; sudan reaction shows lipidic compounds in the outer periclinal walls of ordinary epidermal cells, but not on the walls of ray cells. E. Detail of sclereid ends, showing their connections with vascular bundles (at both ends, left and right); note lateral connections between a sclereid and palisade parenchyma cells. Abbreviations: Asterisks (*) point to ray cell wall. Arrows point to pitted walls. Circles indicate the lateral connections between sclereid and palisade parenchyma cells. Ct, cuticle; Cu, cushion structure at the base of the emergence ray; IR, inner region; LR, lateral ray; Nu, nuclei; OR, outer region; PR, porrect ray; Sc, sclereid. ScE, sclereid end; VB, vascular bundle. N.C. Vitarelli et al. Flora 281 (2021) 151878 7 epidermal one. We also observed morphologies that were intermediate between idioblast and trichome (Figs. 6H-L). We highlight that these distinct cell morphologies were completely mature and did not correspond to early ontogenetic stages. In other words, completely mature secretory structures showed different shapes and possessed intermediate characters between idioblast and trichome cells (Figs. 6A, 6B and 6H-K). Secretory trichomes – In Croton mollis, secretory trichomes occurred on the abaxial leaf surface as unicellular structures. They are formed by a secretory head and a short stalk (Figs. 3G; 6E indicated by arrow, and 6L). 4. Discussion 4.1. Croton mollis and the igap´ o environment Individuals of the studied population of C. mollis dominate the site on the banks of the Rio Negro. In Central Amazonia, where this population is located, the prevailing floodplain forests are differentiated into nutrient-rich white-water v´ arzea and nutrient-poor black/clear-water igap´ o (Prance, 1979). Igap´ o flooded forests occur throughout the main Rio Negro channel and most of its black/clear-water tributaries drain the Precambrian shields of the Guayana Region (Huber et al., 1994) and Fig. 5. Extrafloral nectaries, colleters and laticifers in mature leaves of Croton mollis. A - D. Scanning electron microscopy image of basilaminar nectaries. E. Cleared leaf showing the basilaminar nectaries and marginal colleters. F. Longitudinal section of a basilaminar nectary; note laticifers (arrow), jointly with the vascular system, near the secretory region. G. Cleared leaf showing a marginal nectary. H. Scanning electron microscopy image of a marginal colleter. I. Cleared leaf showing a marginal colleter. J. Longitudinal section of a marginal colleter. K. Articulated laticifers associated with the leaf vascular system. Abbreviations: Arrows point to laticifers. Arrowheads point to idioblasts containing druse-type crystals. Co, marginal colleters; EFN, extrafloral nectary. N.C. Vitarelli et al. Flora 281 (2021) 151878 8 Central Brazil (Wittmann et al., 2010). The floodplains of igap´ os are characterized by impoverished substrates and low primary productivity (Furch, 1997). These igap´ o areas are affected by a long and predictable flood pulse subjecting the vegetation to extended inundation periods of up to 8 months and to flooding amplitudes of up to 10 m or more (Junk, 1989). This dynamic imposes a strong impact on plant assemblages whose species composition and structural patterns are continuously changing along the river channel (Rosales et al., 2001; Wittmann et al., 2004; Albernaz et al., 2012; Montero et al., 2012; Montero and Latrubesse, 2013). Wetland habitats are considered strong environmental filters, selecting individuals and species that can tolerate recurrent inundation and drought during their lifespan, and it is very likely that Amazonian wetland species have evolved into a particularly filtered species pool (Luize et al., 2018). The studied C. mollis population, growing on a rock outcrop and under recurrent inundation, fits the slow stream river rheophytic category sensu van Steenis (1932, 1978). Given the great morphological diversity and wide distribution of Croton in the Neotropics one might expect that the rheophytic habit could have evolved several times, especially in those clades with species inhabiting vast and complex fluvial systems and wetlands such as the Amazon basin (e.g., Riina et al., 2010). There appears to be a relationship between the morphology of the studied individuals of C. mollis and the water level variation, which is suggested by the presence of etiolated branches with nodes and leaves concentrated at the branch tips (Fig. 1B). We speculate that the flooding conditions induce certain reduction in sunlight for the plants or plant organs under water, which could trigger an etiolated growth of branches in order to keep the critical parts of the plant above the water level as long as possible. In the rainy season, C. mollis has one key adaptation, namely the stenophyllous leaves, which are long and narrow with a foliar index (length:width ratio) ≧ 3 (van Steenis, 1978). Stenophyllous Fig. 6. Morphology and distribution of secretory idioblasts and secretory trichomes in Croton mollis. A, B. Front view of secretory idioblasts on adaxial leaf surface (A, Scanning electron microscopy; B, cleared leaf). C - L. Leaf transverse section under light microscopy. C. High density of secretory idioblasts in the mesophyll (asterisk); note secretory trichomes in the abaxial leaf surface (arrows). D. Secretory idioblasts interspersed with palisade parenchyma cells, oriented towards the adaxial leaf surface (asterisk) and smaller secretory idioblast with part of the cell in contact with the adaxial leaf surface (arrowhead). E. Unicellular epidermis; note amphistomatic leaf, secretory idioblast across the mesophyll as part of both palisade and spongy parenchyma (asterisk); secretory idioblast at the border of the mesophyll, but with part of the cell in contact with the external adaxial leaf surface (arrowhead), and secretory trichome from abaxial leaf epidermis (arrow). F. Secretory idioblast interspersed with spongy parenchyma cells, oriented towards the abaxial leaf surface. G. Secretory idioblast merged with spongy parenchyma; note part of the cell reaching the external abaxial leaf surface. H - L. Morphological transition series between secretory idioblast and secretory trichome; note (in H-J) the structure resembling an idioblast and (in K and L) the structure resembling a secretory trichome. Abbreviations: Asterisks (*) point to secretory idioblasts. Arrows point to secretory trichomes. Arrowheads point to secretory idioblasts transitional position between the mesophyll and the epidermis oriented towards the adaxial leaf surface. White circles point to stomata. N.C. Vitarelli et al. Flora 281 (2021) 151878 9 leaves are present in species belonging to several Croton clades (Lima and Pirani, 2008; van Ee and Berry, 2011; Rumeu et al., 2016). In the context of igap´ os, stenophyllous leaves allow a hydrodynamic performance, avoiding ripping by water movement during flooding periods as described for many typical rheophytic species (van Steenis 1952, 1981; Kato and Imaichi, 1992; Nomura et al., 2006; Arioli et al., 2008; Voltolini et al., 2009). However reduction of leaf size is also a common features of plants growing in dry habitats (Givnish, 1979; McDonald et al., 2003). Although speculative, we hipotetize that the presence of stenophyllous leaves in C. mollis could be advantageous under extreme environmental conditions such as flooding (rainy season) and soil water availability (dry season). Although it is more common to find rheophytes growing in streambeds along their margin below flood level, they are not hydrophytes (van Steenis, 1978) and could jointly exhibit hydromorphic and xeromorphic features (Arioli et al., 2008; Voltolini et al., 2009; Lobo et al., 2013). Anatomically, the leaves of C. mollis do not present any obvious adaptation to flooding, such as aerenchyma tissue with loose fitting cells. The mesophyll is very compact, resembling the leaf organization of other Croton species occurring in other habitats (Soares, 2013; Vitarelli et al., 2015, 2016; Feio et al., 2016, 2018) and, as described above, its leaf anatomical features are likely related to the igap´ o environmental stresses during the dry season. A relevant adaptation to the igap´ o environment is the dense leaf indumentum of Croton mollis. The presence of both trichomes and trichome-like emergences on the leaves, along with the presence of etiolated branches with clustered leaves at the apex, and stenophyllous leaves, seems to be implicated in the water balance allowing this species to survive as a rheophyte. According to van Steenis (1978), true rheophytes live their whole life-cycle in the streambed within flood level parameters. Most rheophytes are either perennial herbs or small bushes or shrubs, flowering and fruiting in the short dry period. Some morphological features of C. mollis suggest that it is a perennial rheophyte because it is able to cope with the environmental stress of both the dry and rainy seasons (i.e., unflooded and flooded situations). In fact, most herbarium specimens were collected (all fertile plants) during the dry season (Table A1). In addition, the environmental variables contributing the most to the SDM of C. mollis (such as the precipitation of the coldest quarter and the driest month, along with oxygen available to roots; Table 1) are also linked to habitat preferences common in most rheophytes. The SDM analysis, based on both climatic and edaphic variables, indicates the seasonally floodplain forests and open vegetation along nutrient-poor black or clear-water rivers in the Amazon basin as suitable areas for C. mollis. As expected for a region with uneven botanical exploration like the Amazonian region, some of the suitable areas revealed by the model lack C. mollis herbarium records. The vicinities of Bel´ em (Par´ a) and the mouths of Tapaj´ os and Madeira rivers might be worth exploring for the presence of this species, since these areas are also included in the map of white-sand ecosystems (WSE) of Adeney et al. (2016). Historically, the scrubby vegetation and short-statured forests of WSE have not been considered a high conservation priority, but this will need to change since more studies are pointing out these ecosystems worth conservation efforts due to their unique flora, singular ecological processes, high sensitivity to human disturbance, and overlapping with indigenous lands (Adeney et al., 2016). 4.2. Evidences for the role of emergences in the Amazonian Croton mollis The stellate emergences on the adaxial leaf surface of C. mollis are very similar to the fasciculate type described by Vitarelli et al. (2016) for C. splendidus Mart. ex. Colla (C. section Lamprocroton (Müll.Arg.) Pax in Engl. & Prantl) from the Brazilian highland rock outcrops (campos de altitude) in the Atlantic Rain Forest. These campos de altitude, adjacent to cloud forests, benefit from the presence of fog which provides an important source of atmospheric water (Aparecido et al., 2018). Vitarelli et al. (2016) demonstrated that these emergences have a function related to atmospheric water absorption. Such function could also be attributed to the stellate emergences found in C. mollis, since they are as structurally complex as those in C. splendidus. In C. mollis, the rays cells are also potentially permeable to water entrance, given the presence of cuticle covering these cells was not identified by the sudan test in light microscopy, which could indicate that the cuticle is extremely thin or that it is not present in the ray cells. Besides that, although their cell walls are thick, they are not lignified and have obvious lamellations. These traits are favourable for water absorption from atmospheric moisture. According to Fahn (1990) the lamellation seen in the secondary wall is often the result of the different density of the fibrils that consist of two continuous interpenetrating systems, one of which is formed by cellulose fibrils and the other by microcapillary spaces. These spaces may be filled with lignin, cutin, suberin, hemicellulose, other organic substances, mineral crystals, and, in fresh tissue, also with aqueous solution. The histochemical tests done in the leaves of Croton mollis discard the presence of lipidic substances in the ray cell wall as well as lignin in their main extension. It is likely that the atmospheric moisture penetrates into the ray cell wall by capillarity and the aqueous solution flows into the rays via apoplastic pathway, using the microcapillary spaces of the ray cell wall. The pitted base of rays allows the flow of the aqueous solution toward the interior of the leaf through the sclereids, which maximizes the distribution of water into the mesophyll or vascular bundles. The ability of leaf trichomes to absorb water has been already demonstrated in different plant groups such as Polypodium polypodioides (L.) Hitchc. (Stuart, 1968), various Orchidaceae (Pridgeon, 1981), Microlepis oleifolia (DC.) Triana (Milanez and Machado, 2008), Solanum elaeagnifolium Cav. (Burrows et al., 2013) and Quercus ilex L. (Fern´ andez et al., 2014). Also, in some Bromeliaceae, leaf trichomes are associated to water absorption (Benzing, 1970, 1976; Benzing et al., 1976; Sakai and Sanford, 1980; Brighigna et al., 1988) and they resemble the lepidote trichomes or emergences of some Croton species (Vitarelli et al., 2016), but those Bromeliaceae are structurally different and more complex than the stellate emergences in C. mollis and other congeneric species (Vitarelli et al., 2016). The atmospheric water uptake could be particularly important for Croton mollis, since it grows on sandy soil over rock outcrops. Therefore, although C. mollis occurs in an environment with high annual average rainfall (Central Amazonia), the species appears to be adapted to nutrient-poor sandy soils with low water retention (Prance, 1979; Furch, 1997). According to Oliveira-Filho et al. (2021), in the Amazon region, marginal vegetation types such as the igap´ o undergo eco-physiological stress indicating that substrate, not climate, is the most important environmental driver controlling the major axes of floristic composition in Amazonian plant communities. Igap´ os are found along rivers in drain basins where white sand or other highly leached soils prevail (Oliveira-Filho et al., 2021). Adeney et al. (2016) indicate that the soils upon which these ecosystems occur are acidic and nutrient poor, and aluminum toxicity is important in many locations. In addition, plant communities in some white sand ecosystems must cope with periodic water limitations. While Croton species from the Brazilian campos de altitude appear to take advantage of hydric and nutrition supplements from the daily fogs (Vitarelli et al., 2016), C. mollis might benefit from the high atmospheric humidity of the Amazonian forest as the main source of hydrological input and, hypothetically, an alternative source of nutrients. The presence of emergences in C. mollis supports the idea that these structures are probably more widespread in Croton species occurring in different regions and belonging to different lineages within the genus. Recently, Sodr´ e et al. (2019b) also reported the presence of leaf emergences in a species (C. macrosepalus Sodr´ e & M.J.Silva) of section Geiseleria, but they did not provide any insights into the ecological implications of that finding. Although not indicated with the term emergence, the presence of sclereids connected with leaf trichome-like N.C. Vitarelli et al.