Epidermal micromorphology in Dioon: did volcanismconstrain Dioon evolution?

MARIA ROSARIA BARONE LUMAGA1, MARIO COIRO1*, ELISABETH TRUERNIT2,BOGLÁRKA ERDEI3 and PAOLO DE LUCA1

1Department of Biology, University of Naples ‘Federico II’, I-80139 Naples, Italy2Department of Biology, ETH Zurich, 8008 Zurich, Switzerland3Botanical Department, Hungarian Natural History Museum, POB 222, 1476 Budapest, Hungary

Received 29 October 2014; revised 17 June 2015; accepted for publication 24 July 2015

The genus Dioon occupies an important phylogenetic position as sister to the other Zamiaceae. However, itsepidermal morphology is still poorly known. We employed scanning and transmission electron microscopy, confocalmicroscopy and light microscopy to examine the epidermal and cuticular morphology of 12 of the 14 currentlyrecognized species of Dioon, examining cultivated plants and herbarium specimens. Epidermal characters separateDioon quite clearly from the other genera of the cycads. Within the genus, the major subgroups can bedistinguished by their epidermal morphology. Some characters, such as the degree of stomatal protection, reflectecological specializations in species within the subgroups. Several epidermal characters of the crown group Dioonseem to be absent in the Cenozoic fossils usually associated with the genus, which have been used as calibrationsin many recent molecular dating analyses. The presence of similar characters in a Mesozoic fossil adapted tovolcanic stress offers a new key in the interpretation of Dioon evolution. © 2015 The Linnean Society of London,Botanical Journal of the Linnean Society, 2015, ••, ••–••.

ADDITIONAL KEYWORDS: cuticle – cycads – electron microscopy – palaeobotany – stomata.

INTRODUCTION

The genus Dioon Lindl., originally associated with thetribe Encephalarteae (Stevenson, 1992), is currentlyunderstood to be a clade that diverged from otherZamiaceae in the Cretaceous and differentiated in theMiocene (Nagalingum et al., 2011; Salas-Leiva et al.,2013; Condamine et al., 2015) (Fig. 1). Althoughstudies on the morphology of the genus have beenconducted since Chamberlain (1909), many species inthis genus have only been discovered in more recenttimes and have been less thoroughly studied (Sabato& De Luca, 1985).

Various studies have been carried out in the past onthe epidermal morphology and stomatal structure ofcycads using optical microscopy (Thomas & Bancroft,1913; Florin, 1933; Pant & Nautiyal, 1963; Greguss,1968; Mickle et al., 2011; Griffith, Magellan &

Tomlinson, 2014; Pérez-Farrera, Vovides & Avendaño,2014), but Dioon spp. were poorly represented. Indeed,of the 14 species accepted at present (World List ofCycads, http://cycadlist.org, 2014), only three wereknown in 1933 and four in the 1960s. Moreover,because of the presence of a sclerenchymatous epider-mis and hypodermis, it is difficult to obtain cleancuticles without the destruction of stomatal structure.Consequently, micromorphological characters in Dioonare still poorly known. Nevertheless, these characterscould be potentially useful to shed light on the affinitywith the fossil forms assumed to be related to modernDioon and to clarify the phylogeny of the genus.

Indeed, concerning the two main clades recognizedin the genus on the grounds of nuclear and plastidDNA sequence data (Moretti et al., 1993; Bogler &Francisco-Ortega, 2004; González et al., 2008;Nagalingum et al., 2011; Condamine et al., 2015), thesupport for the Spinulosum clade (D. mejiae Standl. &L.O.Williams, D. spinulosum Dyer ex Eichler andD. rzedowskii De Luca, Sabato & Vázq.Torres) is solid,

*Corresponding author. E-mail: mar.coiro@gmail.com,mario.coiro@systbot.uzh.ch

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Figure 1. Tree representing the relationship of Dioon with the other genera of Cycadales.

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but the position of D. edule Lindl. is poorly defined.Some authors include D. edule in a clade that is sisterto the Spinulosum clade (Moretti et al., 1993;Condamine et al., 2015). González et al. (2008)assigned D. edule (with D. angustifolium Miq.) to aseparate clade allied to the Spinulosum clade andplaced the other Dioon spp. in a Purpusii clade. At thesame time, the relationships in the Purpusii clade areunresolved.

The aim of this study was to analyse the epidermis,stomata and cuticle structure in Dioon spp. to deter-mine whether variation in cuticle micromorphologyreflects current phylogenetic relationships among thespecies. These data were compared with informationavailable in the literature with regard to fossil taxafrom the Mesozoic and Cenozoic which have beenrelated to modern Dioon (Florin, 1933; Horiuchi &Kimura, 1987; Hill & Pole, 1994; Archangelsky et al.,1995; Erdei, Akgün & Barone Lumaga, 2010; Erdei,Manchester & Kvacek, 2012; Tang, 2012), with theaim of tracking the persistence of ancestral charac-ters in the studied species and inferring ecologicalconstraints that acted in early Dioon evolution.

MATERIAL AND METHODSPLANT MATERIAL

Twelve of the 14 species described to date were studied(Table 1). The two species not included are D. argen-teum T.J.Greg., Chemnick, Salas-Mor. & Vovides(Gregory et al., 2003) and D. stevensonii Nic.-Mor. &Vovides (Nicolalde-Moerjón et al., 2009). Samples werecollected from specimens kept in the Herbarium Nea-politanus (Index Herbariorum code NAP; data onherbarium sheets and collection locality are reportedin Table 1, abbreviation HN) and/or from plants culti-vated at the Botanical Garden of Naples (NAP inTable 1). Two herbarium samples collected from twolocalities distant from each other were studied forD. edule and D. sonorense (De Luca, Sabato & Vázq-.Torres) Chemnick, T.J.Greg. & Salas-Mor.

Terminology according to Florin (1933), Pant &Nautiyal (1963), Greguss (1968) and Fryns-Claessens& Van Cotthem (1973) was adopted for descriptions ofepidermal anatomy, including stomatal complexes.

TRANSMISSION ELECTRON MICROSCOPY (TEM)

Leaflet samples of D. edule and D. caputoi De Luca,Sabato & Vázq.Torres, as representative of the twomain clades, were fixed in 3% (v/v) glutaraldehyde in0.1 M phosphate buffer at pH 7.2 overnight at 4 °C,post-fixed in 1% (w/v) osmium tetroxide (OsO4) over-night at 4 °C and dehydrated through an ethanolseries. They were then embedded in Spurr’s resin and

sections were cut at 70 nm using a Reichert–JungSupernova ultramicrotome. Sections were collected on200-mesh uncoated copper grids, stained for 12 min inuranyl acetate, post-stained for 8 min in lead citrateand examined using a FEI/Philips EM 2088 transmis-sion electron microscope at an accelerating voltage of80 kV (Sezione di Microscopia LaMMEC del CeSMA,Università degli Studi di Napoli ‘Federico II’, Naples,Italy).

SCANNING ELECTRON MICROSCOPY (SEM)

For the observation of epidermal micromorphologyand epicuticular waxes, leaflet samples of 12 Dioonspp. (Table 1) were fixed in formalin–acetic acid–alcohol (FAA) (10 : 5 : 50) or preserved in 50%ethanol, dehydrated in a graded ethanol series andcritical point dried in liquid CO2. Otherwise leafsamples were directly air dried. For the observation ofthe cuticle surface, isolated cuticles of D. edule andD. spinulosum were prepared by treating sampleswith 20% Cr2O3 (Alvin & Boulter, 1974) for up to 72 h.All samples were coated with gold to c. 30 nm. Speci-mens were observed under a FEI Quantas 200 envi-ronmental scanning electron microscope (ESEM) atan accelerating voltage of 25 kV (Sezione di Microsco-pia LaMMEC del CeSMA, Università degli Studi diNapoli Federico II, Naples, Italy).

EPIFLUORESCENCE MICROSCOPY

Hand-cut sections of 50% ethanol-fixed leaflet seg-ments of D. edule, D. mejiae, D. purpusii Rose, D. hol-mgrenii De Luca, Sabato & Vázq.Torres andD. califanoi De Luca. & Sabato were mounted inwater and observed with a Zeiss Axio Imager 2 epif-luorescence microscope using a 4′,6-diamidino-2-phenylindole (DAPI) filter for excitation and emission.

CONFOCAL MICROSCOPY

Leaflet sections of D. purpusii, D. holmgrenii, D. rze-dowskii and D. mejiae, as representative of the twomain clades, were fixed in 50% ethanol and preservedat 4 °C. They were then hand sectioned and cleared in1% sodium dodecylsulphate (SDS) and 200 mM NaOHfor up to 1 week and treated with a modified pseudo-Schiff propidium iodide staining solution (Truernitet al., 2008). Briefly, tissues were washed three timesin deionized water and transferred to 1% periodic acidfor 40 min. After rinsing again three times, they weretransferred to Schiff reagent with propidium iodide(100 mM sodium metabisulphite and 0.15 M HCl; pro-pidium iodide to a final concentration of 100 mg mL−1

was freshly added) and stained overnight. Thesamples were then rinsed again and transferred

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through an ethanol series (30%, 50%, 70%) to 1% acidalcohol (1% 5 M HCl in 70% ethanol) for 10–15 min.They were then cleared and mounted according toTruernit et al. (2008). Samples were then observedusing a Zeiss LSM 780 confocal microscope with405 nm excitation and a DAPI emission filter.

LIGHT MICROSCOPY

Leaflet samples of D. edule and D. caputoi were fixedin 3% (v/v) glutaraldehyde in 0.1 M phosphate bufferat pH 7.2 overnight at 4 °C, post-fixed in 1% (w/v)osmium tetroxide (OsO4) overnight at 4 °C and

Table 1. Species examined

Species SourceSpecimen sheet and collectionlocality Distribution and habitat

Dioon angustifolium Miq. HN 14.2 (Linares – 900 m) Tamaulipas and Nuevo Leon,Mexico. Dryer types of deciduousforest, 200–1500 m

Dioon califanoi De Luca & Sabato HN 13.2 (Caltepec – 2000 m) Oaxaca and Puebla, Mexico. Steep,shady slopes in tropicaldeciduous forest, 1800–2350 m

Dioon califanoi NAPDioon caputoi De Luca, Sabato &

Vázq.TorresHN 12.9 (Caltepec – 2000 m) Puebla, Mexico. Dry habitat, steep

slopes in stunted forest, 2000 mDioon caputoi NAPDioon edule Lindl. HN 23.2 (San Luis Potosí – 800 m) Veracruz, Querétaro, San Luis

Potosí, Tamaulipas, Mexico.Tropical deciduous forest andoak forest, 0–1500 m

Dioon edule HN 25.1 (Ciudad Valles – 800 m)Dioon edule NAPDioon holmgrenii De Luca, Sabato

& Vázq.TorresHN 30.9 (San Gabriel Mixtepec –

700 m)Oaxaca, Mexico. Humid forest,

650–850 mDioon holmgrenii NAPDioon mejiae Standl. &

L.O.WilliamsNAP Honduras, Nicaragua. Tropical

broad-leaf forest between 120and 1000 m

Dioon merolae De Luca, Sabato &Vázq.Torres

HN 22.5 (cult. Cintalapa, Oaxaca –600 m)

Chiapas, Oaxaca, Mexico. Tropicalsemi-deciduous forest,800–1200 m

Dioon merolae NAPDioon purpusii Rose HN 1.7 (Barranca el Leon – 1300 m) Oaxaca, Mexico. Tropical deciduous

forest, 1300 mDioon purpusii NAPDioon rzedowskii De Luca, Sabato

& Vázq.TorresHN 31.9 (San Bartolomé Ayautla –

450 m)Oaxaca, Mexico. Crevices of

limestone cliffs, full sun,650–850 m

Dioon rzedowskii NAPDioon sonorense (De Luca, Sabato

& Vázq.Torres) Chemnick,T.J.Greg. & Salas-Mor.

HN 21.4 (Sierra de Alamos –950–1100 m)

Sonora, Mexico. Steep dry slopes,620–1200 m

Dioon sonorense HN 19.3 (Mazatlán – 900 m)Dioon spinulosum Dyer ex Eichler NAP Mexico. Tropical evergreen forest,

20–300 mDioon tomaselli De Luca, Sabato &

Vázq.TorresHN 2.2 (Tecolotlan – 2000 m) Guerrero, Michoacán, Jalisco,

Nayarit, Durango, Mexico.Woodlands, 600–1850 m

Information for distribution and habitat from Jones (2002) and Haynes & Bonta (2007). HN, Herbarium Neapolitanus;NAP, Botanical Garden of Naples.

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dehydrated through an ethanol series. They werethen embedded in Spurr’s resin. Sections, 1.5 μmthick, were stained with 0.05% toluidine blue in dis-tilled water (Berlyn & Miksche, 1976) and observedwith a Zeiss Axiolab microscope.

CHARACTER OPTIMIZATION

Character evolution was studied using Mesquiteversion 3.02 (Maddison & Maddison, 2015). Ancestralstate reconstruction using parsimony was employedon the trees of González et al. (2008) and Morettiet al. (1993). The characters used are presented inTable 2.

RESULTS

The observation of transverse sections of leaflets andcuticles with light, epifluorescence and confocalmicroscopy, and SEM and TEM, allowed us to confirmthe characters already observed in previous studies(Thomas & Bancroft, 1913; Florin, 1933; Pant &Nautiyal, 1963; Greguss, 1968) and to acquire moredata on characters still not detected involving theepidermis, hypodermis and stomatal complexes.

Leaflets of all studied species are hypostomatic. Theabaxial epidermis shows stomata arranged in stoma-tal bands between the veins. Such bands are wider inD. mejiae, D. spinulosum and D. rzedowskii. The longaxes of stomata are lined up parallel to the long axisof the leaflet. On the adaxial epidermis, thick- andthin-walled cells are present (Fig. 2A, B). Both areordered in ill-defined rows parallel to the leaflet axisand are elongated or quadrangular in shape (Fig. 2C,D); the thick-walled cells are usually longer and wider(c. 126 × 26 μm); the thin-walled cells are smaller (c.40 × 16 μm). Anticlinal cell walls are straight toslightly sinuous with undulations c. 6 μm widemarked by connections with adjacent cells (Fig. 2C,D). The inner cuticle shows transversely to longitu-dinally oriented granulations (Fig. 2C, D). Where thecell wall is incompletely digested, residuals of cellwalls show the correspondence of undulation with the

point of tight adhesion to adjacent cells (Fig. 2C, D).Underneath the epidermis, there are two layers ofsclerenchymatous hypodermis (Fig. 2A, B).

On the abaxial epidermis, thick- and thin-walledcells are present, and oxalate druses are present inthe epidermis (Fig. 2E) and mesophyll. Stomata,according to the Fryns-Claessens & Van Cotthem(1973) terminology, are of polyperigenous type (adultstomata with several subsidiary cells, arranged in oneor more rings) and of tetracyclic subtype (Fig. 2E, F),i.e. one ring of subsidiaries and three rings of encir-cling cells (Kranzzellen according to Florin, 1933 ter-minology). The papillae of encircling cells showstriations when observed with light microscopybecause of the presence of cell wall stratification(Fig. 2E). Stomatal complexes are frequently adjacentwithout intervening epidermal cells, stomata are ori-ented with the main axes parallel to the long axis ofthe leaflet and long polar extensions of the cuticle(length c. 15 μm) are present, marking the polarboundaries of the guard cells (Fig. 2G, H).

The studied species show noticeable differences inother epidermal characters on both adaxial andabaxial surfaces.

DIOON ANGUSTIFOLIUM

The abaxial epidermis above the veins shows regu-larly spaced sunken trichome bases (Fig. 3A, B).Stomata are surmounted by papillae of the moreexternal encircling cells, which are partially fusedand delimit the stomatal pit, which is, in part,occluded by papillae produced by intermediate encir-cling cells (Fig. 3B). The adaxial epidermis shows astriated cuticle with striations oriented parallel to thelong axis of the leaflet and flanked by wax agglomer-ate and papillae (Fig. 3C). All surfaces are covered bygranular wax (Fig. 3A–C).

DIOON EDULE

This species shares characters with D. angustifoliumwith regard to trichome bases, arrangement ofstomata, papillae on encircling cells and granular wax(Fig. 3D–F). When it is possible to observe the dorsalsurfaces of the stomata, each shows grooves (width c.2 μm) parallel to the stomatal pore (Fig. 3E). Theadaxial epidermis shows cuticular striations, sparsepapillae and rare trichome bases (Fig. 3F).

DIOON PURPUSII

The abaxial epidermis shows trichomes above theveins and stomatal bands densely covering thestomata (Fig. 4A, B). Stomata are surmounted bypapillae of the more external encircling cells, which

Table 2. Characters used in the character optimizationanalysis

Number Character Character states

1 Stomatal pit 0 protected; 1 open2 Trichome bases 0 present; 1 absent3 Trichome base type 0 non-sunken; 1 sunken4 Encircling cell papillae 0 absent; 1 present5 Stomatal bands 0 wide; 1 narrow6 Adaxial cuticle 0 striate; 1 smooth

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are partially fused and delimit the stomatal pit. Theadaxial epidermis shows a smooth cuticle with faintstriae and sparse trichome bases (Supporting Infor-mation, Appendix S1).

DIOON MEROLAE

The stomatal complexes are roughly arrangedobliquely–transversely to the leaflet axis with stomataoccasionally sharing a common pit (Fig. 4C). Stomataare surmounted by papillae of the more external

encircling cells; these are partially fused and delimitthe stomatal pit (Fig. 4C). Papillae produced by theouter and intermediate encircling cells occlude par-tially the stomatal pit (Fig. 4C). The adaxial epidermisshows faintly present cuticular striae and rare tri-chome bases (Fig. 4D).

DIOON CAPUTOI

The stomatal complexes are arranged obliquely ortransversely to the leaflet axis (Fig. 4E). Stomata are

Figure 2. Dioon edule (A, B, E, F): A, B, adaxial side of the leaflet section showing cuticle (c), epidermis (ep), hypodermis(hy) and palisade layer (pl); E, F, abaxial side of the leaflet section showing cuticle (c), epidermis (ep), hypodermis (hy),mesophyll (m), papilla (p) and guard cells (gc). Dioon spinulosum (C, D, G, H): C, D, adaxial cuticle, cell wall undulation(asterisk); G, H, abaxial cuticle, guard cell polar extensions (asterisk). A, E, Light microscopy; B, F, epifluorescencemicroscopy; C, D, G, H, scanning electron microscopy (SEM). Scale bars, 50 μm (A–F); 100 μm (G); 10 μm (H).

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surmounted by papillae of the more external encir-cling cells; these are partially fused and delimit thestomatal pit (Fig. 4E). Papillae produced by the inter-mediate encircling cells almost completely occlude thestomatal pit (Fig. 4E). The adaxial epidermis shows asmooth cuticle (Fig. 4F).

DIOON HOLMGRENII

The abaxial epidermis shows stomata arranged instomatal bands in areas between the veins (Fig. 5A).Stomata are surmounted by a stomatal pit of irregu-lar shape (Fig. 5A); small papillae are formed by the

intermediate encircling cells (Fig. 5A). The adaxialepidermis shows a faintly striate cuticle with scarcetrichome bases (Fig. 5B).

DIOON CALIFANOI

The stomatal complexes are arranged obliquely ortransversely to the leaflet (Fig. 5C). Stomata are sur-mounted by papillae of the more external encirclingcells which partially fuse and delimit the stomatal pit(Fig. 5C, D). The latter are partially occluded bycrypts produced by the encircling cells (Fig. 5C, D).The adaxial epidermis shows papillae (Appendix S1).

Figure 3. Dioon angustifolium (A–C): A, B, abaxial epidermis showing sunken trichome bases (tb), stomatal bands andstomatal pits with papillae (pa); C, adaxial epidermis showing striations, papillae (asterisk) and wax agglomerate (wa).Dioon edule (D–F): D, E, abaxial epidermis showing sunken trichome bases (tb), stomatal bands and stomatal pits withpapillae (pa); F, adaxial epidermis showing striations and papillae (asterisk). A–F, scanning electron microscopy (SEM).Scale bars, 100 μm (A, C, D, F); 50 μm (B, E).

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DIOON SONORENSE

The stomatal complexes are arranged transverselyto the leaflet; this alignment allows the formation ofcrypts enclosing more stomata (Fig. 5E). Thestomata are almost completely occluded by papillaeproduced by the intermediate encircling cells andsurmounted by papillae of the more external, par-tially fused encircling cells which delimit the stoma-tal pit. This is virtually occluded by stomatal cryptsproduced by external encircling cells and covered byepicuticular wax (Fig. 5E). The adaxial epidermisshows an almost smooth cuticle and rare trichomes(Appendix S1).

DIOON TOMASELLII

Stomata are surmounted by a stomatal pit of irregu-lar shape produced by external encircling cells; papil-lae are present at the opposite side of the stomatal pit(Fig. 5F). The adaxial epidermis shows a faintlystriate cuticle (Appendix S1).

DIOON MEJIAE

The stomatal complexes are coarsely arranged trans-versely to obliquely to the long axes of the leaflet(Fig. 6A). Stomata are surmounted by papillae ofthe more external encircling cells, which are fused

Figure 4. Dioon purpusii (A, B): A, B, abaxial epidermis showing trichomes and stomatal pits with papillae (pa); trichomebases (tb) are also visible. Dioon merolae (C, D): C, abaxial epidermis showing stomatal bands and stomatal pits withpapillae; D, adaxial epidermis showing trichome bases (tb). Dioon caputoi (E, F): E, abaxial epidermis showing stomatalpits with papillae (pa); F, adaxial epidermis. A–F, scanning electron microscopy (SEM). Scale bars, 100 μm (A, C, D, E,F); 50 μm (B).

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and delimit the stomatal pit (Fig. 6A). The adaxialepidermis shows a smooth, faintly striated cuticleand rare trichomes (Supporting Information,Appendix S2).

DIOON SPINULOSUM

The stomatal complexes are poorly ordered trans-versely to obliquely to the long axes of the leaflet(Fig. 6B). Stomata are surmounted by papillae ofthe more external encircling cells, which are par-tially fused and delimit the stomatal pit (Fig. 6B).The adaxial epidermis shows a smooth cuticle(Appendix S2).

DIOON RZEDOWSKII

The stomatal complexes are arranged transverselyto the long axes of the leaflet (Fig. 6C). Stomata aresurmounted by papillae of the more external encir-cling cells, which are fused and delimit the stomatalpit (Fig. 6C); trichome bases appear in stomatalbands (Fig. 6D). The adaxial epidermis shows afaintly striated cuticle and papillae (Appendix S2).

Confocal microscopy (Fig. 7A–H) and TEM(Fig. 8A–D) show the different development of papil-lae in the studied species (Fig. 7A–E). Papillae areformed by expansions of intermediate encircling cells(length c. 5 μm in D. purpusii, 3 μm in D. rzedowskii)and by the external encircling cells [length c. 30 μm in

Figure 5. Dioon holmgrenii (A, B): A, abaxial epidermis showing stomatal bands with shallow stomatal pits; adaxialepidermis showing scarce trichome bases (tb). Dioon califanoi (C, D): C, D, abaxial epidermis showing crypts (cr) andpapillae (pa). Dioon sonorense: E, adaxial epidermis showing crypts (cr). Dioon tomasellii: F, abaxial epidermis showingpapillae (pa). A–F, scanning electron microscopy (SEM). Scale bars, 100 μm.

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D. purpusii (Fig. 7A) and almost absent in D. hol-mgrenii (Fig. 7B)].

The cuticular membrane is of substantial thickness(c. 3–5 μm) on the abaxial side of the epidermis and c.3–7 μm on the adaxial side (Figs 2A, B, E, F, 7A–H,8A–D). The cuticular membrane is formed by thecuticle proper or layer A (c. 1–2 μm) and the cuticularlayer or layer B (c. 2–5 μm); in the case of papillae, itis possible to detect in the cuticle proper (layer A) thepresence of a lamellated A1 layer about 150–200 nmthick and an inner homogeneous A2 layer (Fig. 8A, B).The cuticle of the pavement cells does not show theA1 layer (Fig. 8D).

Papillae formed by external encircling cells arealmost absent in D. holmgrenii (Fig. 7B), which alsoshows a reduction in the number of thick-walledencircling cells. Dioon mejiae presents a similarreduction of papillae and number of thick-walledencircling cells, thus resulting in a shallow pit(Fig. 7D). On the contrary, D. califanoi shows thepresence of sunken stomatal pits with a largenumber of thick-walled encircling cells and an epi-dermis with tall cells (Fig. 7C). In D. purpusii, it ispossible to observe the presence of a central bridge-like structure between guard cells of a stoma(Fig. 7A). The trichomes observed in D. purpusii do

not show characters distinctive of secretory struc-tures (Fig. 7H).

CHARACTER OPTIMIZATION

Of the characters used for character optimization,only two did not show homoplasy. These were thepresence or absence of encircling cell papillae (Sup-porting Information, Appendix S3) and the width ofthe stomatal bands (Fig. 9A), which were distributedin the same way among Dioon spp. The difference inthe reconstruction between the trees of Moretti et al.(1993) and González et al. (2008) involves only theancestral state of the character, which is ambiguouson the topology of Moretti et al. (1993). On the tree ofGonzález et al. (2008), the ancestral state for thegenus appears to be narrow stomatal bands andencircling cell papillae present, with the evolution ofwide stomatal bands and the loss of encircling cellpapillae in the ancestor of the Spinulosum clade.

Other characters show high to moderate levels ofhomoplasy. Trichome bases seem to have been lostfour times (in the framework of Moretti et al., 1993)or three or four times (in the framework of Gonzálezet al., 2008). The striation of the cuticle follows asimilar pattern, with three independent losses in both

Figure 6. Dioon mejiae: A, abaxial epidermis showing stomatal bands with shallow stomatal pits. Dioon spinulosum: B,abaxial epidermis showing stomatal bands with shallow stomatal pits. Dioon rzedowskii (C, D): C, D, abaxial epidermisshowing stomatal bands with shallow stomatal pits and trichome bases (tb). SEM. Scale bar, 100 μm (A, B, C); 50 μm (D).

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frameworks. The evolution of the trichome base typeshows pretty clearly that a sunken trichome baseevolved in the ancestor of D. edule and D. angustifo-lium, and such inference is independent of the phy-logenetic framework used (Fig. 9B).

The protection of the stomatal pit follows a peculiarpattern (Fig. 9C): in the framework of Moretti et al.(1993), the character at the base of the tree is ambigu-ous and the presence of open pits in D. holmgrenii canbe interpreted as either a parallelism with the Spi-

nulosum clade or a reversal; in the framework ofGonzález et al. (2008), a parallel evolution of open pitsis the preferred hypothesis.

DISCUSSIONEVOLUTION AND PHYLOGENETIC DISTRIBUTION OF

EPIDERMAL CHARACTERS IN DIOON

In Dioon, the micromorphological characters of theepidermis appear to be quite stable within species.

Figure 7. Dioon purpusii (A, H); D. holmgrenii (B); D. califanoi (C); D. mejiae (D, F); D. rzedowskii (E, G), A–E, Abaxialepidermis showing guard cells (gc), subsidiary cells (sc), encircling cells (ec), papillae (pa), cell wall (cw), cuticular layerA (cA) and cuticular layer B (cB); F–H, adaxial epidermis showing cell wall (cw), cuticular layer A (cA), cuticular layerB (cB) and trichome base (tb). A, B, D–H confocal microscopy; C, epifluorescence microscopy. Scale bars, 10 μm.

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The structure of the stomatal complex, as shown byfluorescence and confocal microscopy, is characteristicfor this genus: it is possible to observe one layer (orrarely two) of subsidiary cells with thin walls, andthree (rarely two) layers of thick-walled encirclingcells, which resemble normal epidermal cells. Thissets Dioon apart from other cycads with deep stoma-tal pits, such as Cycas L. section AsiorientalesJ.Schust. (Griffith et al., 2014), in which the pit isformed by the elongation of a single subsidiary and anencircling cell, and some species of EncephalartosLehm. and Macrozamia Miq. (Thomas & Bancroft,1913), in which the number of encircling cells isirregular. This is not surprising in the light of molecu-lar analyses (Salas-Leiva et al., 2013), which indicatethat Dioon is the sister group to all other Zamiaceaeand not particularly related to the Encephalarteae(Stevenson, 1992), and that Cycas with deep pitsevolved from species with shallower pits (Griffithet al., 2014). Dioon also appears to be different fromthe other Mexican cycads in the structure of thestomata. Zamia L. and Ceratozamia Brongn. havequite superficial stomata, with a stomatal pit formedby subsidiary cells only in Zamia (Thomas &Bancroft, 1913; Papadopoulos, 1928; Acuña-Castillo &Marín-Méndez, 2013) and by subsidiaries and onelayer of encircling cells in Ceratozamia (Barone

Lumaga, Moretti & De Luca, 1999; Vovides et al.,2004). The peculiar structure of the stomata of Diooncan be considered as a genuine synapomorphy of thegenus. However, the lack of information on manyspecies from the other genera does not allow us toprecisely reconstruct the evolution of the accessorycell number in Cycadales.

The limited variation among Dioon spp. reflectsthat observed for Cycas (Mickle et al., 2011; Griffithet al., 2014). However, some interesting differencescan still be noted: D. mejiae, D. spinulosum andD. rzedowskii have wider and flatter stomatal bands,with less stomata and shallower, more open stomatalpits. The rest of the species share deeper, more pro-tected stomata (with the exception of D. holmgrenii)distributed more densely in narrower, slightly sunkenstomatal bands. Dioon edule and D. angustifoliumshare the presence of sunken trichome bases.

The reconstruction of the evolution of epidermalcharacters in the genus is complicated by the unre-solved issues regarding phylogenetic relationships.Sabato & De Luca (1985) hypothesized the presence ofthree main evolutionary lines with different ecologicalspecializations, represented by D. mejiae, D. spinulo-sum–D. rzedowskii and the remaining species. Morettiet al. (1993), using plastid DNA restriction fragmentlength polymorphisms (RFLPs) and morphology,

Figure 8. Dioon edule: A–D, transmission electron microscopy (TEM); abaxial epidermis showing cuticular layer A1(cA1), cuticular layer A2 (cA2), cuticular layer B (cB) and cell wall (cw). Scale bars, 1 μm (A); 100 nm (B); 1 μm (C);500 nm (D).

12 M. R. BARONE LUMAGA ET AL.

© 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, ••, ••–••

grouped Dioon spp. into two clades: the xeromorphicEdule clade, corresponding to one evolutionary groupof Sabato & De Luca (1985), and the Spinulosum clade,corresponding to the other two. A similar rooting of the

phylogenetic tree for the genus was obtained byCondamine et al. (2015) in their Bayesian analysis ofCycadales. The analysis of González et al. (2008), usingnuclear internal transcribed spacer (ITS) and plastid

Figure 9. Reconstruction of epidermal characters on the phylogenetic tree of Dioon. A, Stomatal band character. B,Trichome base type. C, Stomatal pit. Left, phylogeny according to Moretti et al. (1993); right, phylogeny according toGonzález et al. (2008).

EPIDERMAL MICROMORPHOLOGY IN DIOON 13

© 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, ••, ••–••

trnL-trnF markers, retrieved a different rooting of thetree, with D. edule and D. angustifolium united assister to the Spinulosum clade. One sample of D. tom-masellii [later identified as a separate species, D. ste-vensonii (Nicolalde-Moerjón et al., 2009)] wasretrieved as sister to this Edule–Spinulosum clade. APurpusii clade was retrieved with strong support, butlow resolution of the relationships between species.

Based on the optimization of our characters in thetwo phylogenetic analyses, our data do not particu-larly support the topology of either González et al.(2008) or Moretti et al. (1993). Even if some charac-ters, such as narrow stomatal bands and encirclingcell papillae, could be interpreted as synapomorphiesfor an Edule clade sensu Moretti et al. (1993), theabsence of a proper outgroup to polarize characterevolution makes the alternative interpretation as ple-siomorphies equally probable. Indeed, the main dif-ference between the topologies of González et al.(2008) and Moretti et al. (1993) lies in the ancestralstate reconstruction of the former two characters andof the stomatal pit protection character. In the frame-work of Moretti et al. (1993), the ancestral state isambiguous for all three characters, compatible withthe hypothesis of Moretti et al. (1993) about an earlyseparation of a mesic lineage (the Spinulosum clade)and a xeric lineage (the Edule clade). In the back-ground of González et al. (2008), the ancestral Dioonis inferred as having protected stomatal pits, narrowstomatal bands and encircling cell papillae, with theevolution of new characters in the Spinulosum clade.

RE-EVALUATION OF THE FOSSIL RECORD OF DIOON

If we compare the micromorphological characters ofthe studied Dioon spp. with fossil taxa traditionallyassociated with the modern genus [Dioonopsis nip-ponica from the Palaeogene of Japan (Horiuchi &Kimura, 1987), Dioonopsis praespinulosa (Hollick)Erdei, Manchester, Kvacek from the Eocene of Alaska(Erdei et al., 2012), Dioonopsis macrophylla (Potbury)Erdei, Manchester, Kvacek from the Eocene of Cali-fornia (Erdei et al., 2012) and Pseudodioon akyoliErdei, Akgün, Barone Lumaga from the Miocene ofTurkey (Erdei et al., 2010)], we notice that none ofthese taxa fits in the micromorphological variationpresent in the extant species. The fossils, althoughthey resemble Dioon on a macromorphological level,share few micromorphological characters with theextant species (Horiuchi & Kimura, 1987; Kvacek &Velitzelos, 2000; Erdei et al., 2010, 2012; Tang, 2012).Fossil leaves of Dioonopsis and Pseudodioon displaymostly isodiametric epidermal cells which are notdifferentiated into thick- and thin-walled cells, pre-sumably less sunken stomata with only one recon-structed accessory cell circle and a cuticular coronal

rim. Moreover, stomata are scattered, not arranged instomatal bands in Dioonopsis spp. Their assignationto the stem of Dioon (Moretti et al., 1993) appears tobe weakly supported by cuticle micromorphology. Thepreservation of the cuticle in Pseudodioon does notallow an appropriate reconstruction of the structureof the stomatal pit (Erdei et al., 2010). Although atransverse section of stomata was provided (Erdeiet al., 2010, fig. 6f), the exact stomatal structure isunclear as a result of heavy compression of thecuticle. Nevertheless, stomatal complexes of Pseudo-dioon display a large number of subsidiary/encirclingcells which are not observable among Dioon spp.Consequently, the data discernible from the abovefossil do not give strong support to their proximity tothe crown group Dioon. Some recent molecularstudies (Crisp & Cook, 2011; Nagalingum et al., 2011;Salas-Leiva et al., 2013; Condamine et al., 2015) usedfossils with Dioon-like macromorphology to constrainthe stem node of the genus to the Palaeocene, but ourdata suggest that macromorphology alone is not suf-ficient to assign a fossil to the Dioon lineage.

A noteworthy example from the Mesozoic, Pseu-doctenis ornata (Archangelsky et al., 1995) from theEarly Cretaceous of Patagonia, shows comparablemicromorphological characters to Dioon. Althoughdisplaying isodiametric epidermal cells and scatteredstomata, also characteristic of the younger Palaeo-cene cycads, this fossil leaf shares characters withDioon spp. from the Purpusii and Edule clades(Table 3), i.e. pit characters, number of accessory celllayers, presence and structure of the trichome basesand presence of longitudinal striations on the adaxialcuticle. Other characters shared are the presence ofthick cuticles, the ultrastructure of the cuticleshowing the presence of pectin visible as electron-dense fibrous material (plate VI, figs 37–40;Archangelsky et al., 1995), epidermis with groups ofthickened cells (plate I, figs 8, 9; Archangelsky et al.,1995) and a ‘barrier’ of papillae covering the stomatalpit with the two lateral papillae more developedthrough the pit (plate III, figs 18, 19, 22–24;Archangelsky et al., 1995). The presence of a centralbridge-like structure, comparable with that in D. pur-pusii (Fig. 6A), was also reported in P. ornata (plateIV, fig. 29; Archangelsky et al., 1995).

STOMATAL STRUCTURE AND ECOLOGY IN DIOON

Some of the morphological traits in Dioon seem to berelated to ecological specializations. The protection ofthe stomatal pit, for example, is reconstructed to haveevolved independently in the Spinulosum clade andin D. holmgrenii (Fig. 9C), which occupies the mostmesic habitat among the members of the Purpusiiclade (Table 1). However, the uncertain phylogeny of

14 M. R. BARONE LUMAGA ET AL.

© 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, ••, ••–••

Tab

le3.

Com

pari

son

ofse

lect

edm

acro

-an

dm

icro

mor

phol

ogic

alch

arac

ters

from

Pse

ud

octe

nis

orn

ata

(Arc

han

gels

kyet

al.,

1995

),D

ioon

opsi

sn

ippo

nic

a(H

oriu

chi

&K

imu

ra,

1987

),D

.pra

espi

nu

losa

(Erd

eiet

al.,

2012

),D

.mac

roph

ylla

(Erd

eiet

al.,

2012

),P

seu

dod

ioon

akyo

li(E

rdei

etal

.,20

10)

and

Dio

on(s

tudi

edsp

ecie

s)

Spe

cies

/ch

arac

ters

Lea

flet

dim

ensi

onE

pide

rmal

papi

llae

Tric

hom

esO

ccu

rren

ceof

stom

ata

Pit

char

acte

r

Nu

mbe

rof

acce

ssor

yce

llla

yers

Cu

ticu

lar

stri

aeon

adax

ial

surf

ace

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xial

epid

erm

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llfe

atu

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avem

ent

cell

anti

clin

alw

all

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viro

nm

enta

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tion

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ent

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onto

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10.0

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0×

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tA

bsen

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llae

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ain

tly

pres

ent

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nga

te,

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ws

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oodl

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tan

otav

aila

ble.

Info

rmat

ion

for

pave

men

tce

llan

ticl

inal

wal

lfr

omE

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.(2

010)

.In

form

atio

nfo

ren

viro

nm

enta

lco

ndi

tion

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seu

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ten

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nat

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.(1

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010)

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002)

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cept

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ejia

efr

omH

ayn

es&

Bon

ta(2

007)

.

EPIDERMAL MICROMORPHOLOGY IN DIOON 15

© 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, ••, ••–••

the genus does not allow us to infer the ancestralecology of Dioon. The reconstruction based on thetopology of González et al. (2008) could imply thatDioon adapted early in its history to arid environ-ments, with characters such as papillae, protectedstomatal pits, and narrow and almost encrypted sto-matal bands, and the ancestor to the Spinulosumclade successively adapted to a more mesic environ-ment. The topology of Moretti et al. (1993), however,implies that Dioon could represent an ancestrallymesic genus which secondarily adapted to more xerichabitats during its diversification.

It is, however, clear that many characteristicsusually linked with xeromorphy are present in allspecies of the genus, i.e. stomatal chambers, scle-rotized hypodermis, thick cuticle, etc. In particular,the peculiar structure of the stomatal apparatusthat allows the guard cells to be sunken well belowthe epidermal level does not show variation amongspecies: the deeper stomatal pit of the more xero-morphic species and the shallower pit of the mesicspecies consist of the same number of encirclingcell strata. Given the distance between Dioonand its closest relatives (Salas-Leiva et al., 2013;Condamine et al., 2015), the hypotheses about theevolution of such characters are highly dependenton the data originating from the fossil record. Thepositioning of Dioonopsis on the stem of Dioonwould imply that the structure of the stomatalcomplex evolved quite late in the genus (after thePalaeogene) and would be associated with the crowngroup Dioon. However, we believe that the supposedsynapomorphies of Dioonopsis and Dioon are ratherweak (Kvacek & Velitzelos, 2000; Erdei et al., 2012;Tang, 2012).

IS THE XEROMORPHIC STRUCTURE OF DIOON A

CONSEQUENCE OF VOLCANISM?

The similarity of stomata between Dioon and Pseu-doctenis ornata offers another key for interpretationof the morphology of the extant species. Pseu-doctenis ornata shared a habitat with conifers,ferns, Bennettitales and few pteridosperms in afluvial valley under mild seasonal climatic condi-tions on a soil formed and developed by a continu-ous volcanic activity with ash deposit, and thecharacters of its cuticle have been interpreted as anadaptation to a volcanically active environment(Archangelsky et al., 1995). Indeed, many characterscommonly associated with xeromorphy, includingwax plugs, thick cuticle, stomatal papillae and tri-chomes, can also be associated with volcanic stress.Indeed, the water repellence/self-cleaning function ofsuch structures reduces the damage from dust/ash and toxic pollutants (Haworth & McElwain,

2008). The most recent molecular dating analyses(Nagalingum et al., 2011; Salas-Leiva et al., 2013;Condamine et al., 2015) place the origin of the stemgroup Dioon during the Cretaceous period, a periodof strong volcanic activity with the origin of manyLarge Igneous Provinces (Courtillot & Renne, 2002).Almost all Dioon spp. are distributed in areas whichwere exposed to continuous volcanic activity behindthe collision belt between the Yucatán Block andMexico in the Miocene (Kim, Clayton & Keppie,2011) and the formation of the Trans Mexican Vol-canic Belt. According to Kim et al. (2011), the vol-canic arc was active along the coast until c. 25 Myaand reappeared during the early Miocene. This over-laps with the inferred age for the diversification ofthe crown group Dioon (Nagalingum et al., 2011;Condamine et al., 2015). An ancestral adaptation tovolcanism could explain some of the issues in theinterpretation of the epidermal morphology of Dioon.The structure of the stomatal complex, with itsthree layers of encircling cells, could represent aresponse to the high level of volcanic activity in theMesozoic, allowing the plant to avoid the occlusionof stomatal pores by volcanic ash and the penetra-tion of toxic gases (Haworth & McElwain, 2008).These characters could then have been helpful inallowing the ancestor of the crown group Dioon topersist in the volcanically active area of Mexico, andcould have been recruited during the recent radia-tion of the genus, allowing the species to persist bycolonizing more xeric environments or shallowersoils (Vovides, 1990; Carpenter, 1991; Gregory &Chemnick, 2004).

According to our hypothesis, the evolution of thepeculiar structure of the stomatal pit of Dioonshould precede the radiation of the genus, and fossilswith Dioon-like micromorphology should be found inpalaeoenvironments that show evidence of volcanicactivity. Pseudoctenis ornata could represent anexample of such a fossil: it shares many micro-morphological characters with Dioon, and its age(114.67 ± 0.18 Mya; Césari et al., 2011) is compatiblefor a position on the stem group of Dioon. However,this fossil lacks many of the characters shared byextant Zamiaceae, and its geographical positionwould imply a southern American origin of Dioonwhich is not currently supported by other sources ofdata (Salas-Leiva et al., 2013). If Dioonopsis should beplaced on the stem group of Dioon (based on dataother than leaflet morphology, such as morphology ofthe cones, etc.), this would imply that the charactersshared by the crown group Dioon evolved after theEocene. In this scenario, an adaptation to the morexeric environments of the late Miocene (Nagalingumet al., 2011) could be the preferred explanation for themorphology of Dioon.

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CONCLUSIONS

Our study underlines the unique structure of thestomatal apparatus in Dioon, which distinguishesthis genus from all other members of Zamiaceae.Moreover, although many characters are invariant ordo not show a clear phylogenetic distribution, wehave identified some potentially useful systematiccharacters (trichome bases in the Edule clade) andsome characters which seem to be associated withhabitat (open or closed stomata). Our new data allowus to conclude that the fossil record of Dioon ishighly incomplete and that many Cenozoic taxa pre-viously associated with this genus need to be recon-sidered. We also advance a new hypothesis about themorphology of the epidermis in Dioon that could berelated to a past adaptation to volcanic stress. Morework is needed to clarify some issues about theecology and evolution of Dioon. In our opinion, amicroevolutionary approach to the variation of epi-dermal morphology in Dioon would be particularlyimportant. This could allow the identification ofmore subtle variation among different populationsand the correlation of this variation with ecology orpopulation structure (Pérez-Farrera et al., 2014).Nevertheless, the investigation of additional fossilsrelated to Dioon, especially in the late Mesozoic,would be crucial to an understanding of the relation-ship between fossil taxa and modern genera and toresolve the biogeographical and evolutionary historyof the group. The many hypotheses advanced usingmolecular data (Crisp & Cook, 2011; Nagalingumet al., 2011; Salas-Leiva et al., 2013) need to be inde-pendently validated using the fossil record. In thislight, a thorough knowledge of epidermal anatomyplays a pivotal role in the understanding of cycadevolution.

ACKNOWLEDGEMENTS

The authors thank the staff of the Naples BotanicalGarden for help with sample collection and, in par-ticular, Dr Roberta Vallariello for obtaining samplesfrom the Herbarium Neapolitanum (NAP) Universityof Naples ‘Federico II’. We also acknowledge the Asso-ciate Editor and an anonymous reviewer for substan-tially improving the manuscript. B.E. is grateful tothe Hungarian Scientific Research Fund for support-ing her studies (OTKA 108664).

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SUPPORTING INFORMATION

Additional Supporting Information may be found in the online version of this article at the publisher’s website:

Appendix S1. Scanning electron microscopy (SEM); adaxial epidermis of Dioon purpusii, D. califanoi, D.sonorense, D. tomaselli.

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Appendix S2. Scanning electron microscopy (SEM); adaxial epidermis of Dioon mejiae, D. spinulosum, D.rzedowskii.Appendix S3. Reconstruction of characters on the phylogeny of Dioon. Phylogeny on the left from Moretti et al.(1993), phylogeny on the right from Gonzàlez et al. (2008). On the top row character ‘Encircling cell papillae,’on the middle character ‘Adaxial cuticle,’ on the bottom character ‘Trichome bases.

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