EUPHORBIACEAE) in RESPONSE to DAMAGE by Urania Fulgens WALK ER

Home , Alcides, Chrysiridia, Claoxylon, Micrococca, Omphalea, Sapium

ANATOMY, HISTOCHEMISTRY AND PHENOLIC COMPOUNDS CONTENT OF LEAVES FROM Omphalea oleifera Hemsl. (EUPHORBIACEAE) IN RESPONSE TO DAMAGE BY Urania fulgens WALK ER

Silvia Espinosa-Matías, Roberto Enrique Llanos-Romero, Álvaro Delfino Campos Villanueva, Blanca Pérez-García, Josefina Herrera-Santoyo and Patricia Guevara-Fefer

SUMMARY

Leaves of Omphalea oleifera Hemsl. damaged by Urania crystals. The estimated amounts of lignin and cafeic, ferulic fulgens Walker were examined. Leaves were anatomically de- and chlorogenic acids were respectively 11% and 109.65, 16.58 scribed, histochemical tests were performed and the content of and 0.082µg·g-1 dry wt in the damaged leaves, whereas for the lignin and phenolic acids estimated. Morpho-anatomical fea- intact were 7%, 97.65, 5.48 and 0.051µg·g-1 dry wt. The results tures were similar in damaged leaves and the control, but mi- suggest that the insect damage triggers induced responses in nor histochemical differences were observed. The tissues adja- O. oleifera including production and accumulation of phenolic cent to the damage showed lignin deposits and calcium oxalate compounds and calcium oxalate crystals on the leaf tissues.

Introducción niidae) primarily feed on stems, mushroom-shaped an- Plants exhibit a wide gamut Omphalea leaves (Lees and droecia, and large fruits (Ru- of induced responses to the Omphalea L. (Euphorbia- Smith, 1991). At the Estación dall, 1994a, b; Gillespie, 1997; damage caused by pathogens ceae) is a genus of canopy li- de Biología Tropical Los Gillespie and Ambruster, and herbivores. Particularly, anas, shrubs and trees, com- Tuxtlas, Veracruz, the larvae 1997). Regarding O. oleifera, the induced responses that prised of ~20 tropical species of Urania fulgens Walker the seedlings content of some currently decrease the nega- with centers of diversity and 1854 feed on O. oleifera to an secondary metabolites (Del tive fitness consequences of endemism in the Caribbean extent that has been consid- Amo et al., 1986), its massive attacks on plants are termed and Madagascar (Gillespie, ered by some authors as an defoliation by U. fulgens and ‘induced defenses’ (Karban 1997; Radcliffe-Smith, 2001). uncommon case of high mag- the presence of peduncular and Balwind, 1997). These The genus is represented in nitude defoliation (Dirzo and extrafloral nectaries, have responses can be morphologi- Mexico by Omphalea oleifera Mota-Bravo, 1997). been reported (Dirzo and cal, chemical or a combina- Hemsl.; individuals are trees Studies of Omphalea have Mota-Bravo, 1997) and the tion of both. There are nu- 25-30m tall that form part of focused on chemistry and tax- character afterwards con- merous examples of constitu- the canopy and sub-canopy of onomy. Kite et al. (1991, 1997) firmed and described (Aguirre tive (always expressed in the the high evergreen tropical reported the presence of alka- et al., 2013). Though it is plant) and chemical plant re- rainforest of the states of loidal glycosidase inhibitors in known that plants as well as sponses (see Karban and Oaxaca and Veracruz (Dirzo the species O. diandra and O. herbivores have developed mo- Balwind (1997) and references and Mota-Bravo, 1997). The queenslandiae, and other au- lecular, physiological or behav- therein). The majority of larval stage of diurnal moths thors have described features ioral adaptations to cope with plants produce phenolic acids of the genera Urania Fa- considered common in the the deleterious effects in their or their derivatives such as bricius, Chrysiridia Hübner genus such as white or red la- relationship (Konno, 2011), the phytoalexins, flavonoids and and Alcides Hübner, all be- tex, nonarticulated laticifers, effects of O. oleifera on the lignin (Harborne, 1988). In longing to the subfamily extrafloral nectaries, liana hab- larvae of Urania fulgens or addition to a structural role, Uraniinae (Lepidoptera: Ura- it with tendril-like climbing viceversa are unknown. lignin confers protection

KEYWORDS / Calcium Oxalate / Histochemistry / Omphalea / Phenolic Acid / Received: 01/08/2015. Modified: 06/20/2016. Accepted: 06/23/2016.

Silvia Espinosa Matías. Doctor in e-mail: enrique.llanos.r@gmail. Universidad Autónoma Metro- versidad Autónoma de Barce- Sciences in Plant Biology, com politana - Iztapalapa, Mexico. lona, España. Professor-Re- Universidad Nacional Autó- Álvaro Delfino Campos Villa- e-mail: [email protected] searcher, UNAM, Mexico. noma de México (UNAM). Lab nueva. Master in Sciences in Josefina Herrera Santoyo. Doc- Address: Phytochemistry La- Technician, UNAM, Mexico. Biology, UNAM, Mexico. Lab tor in Sciences in Biology, boratory, Faculty of Sciences, e-mail: [email protected] Technician, UNAM, Mexico. UNAM, México. Professor, UNAM. Av. Universidad 3000, Roberto Enrique Llanos- e-mail: [email protected] UNAM, Mexico. e-mail: jhs@ C.P. 04510, Coyoacán, Mexico Romero. Master in Sciences in Blanca Pérez-García. Doctor in ciencias.unam.mx City, Mexico. e-mail: patricia- Biology, UNAM, Mexico. Lab Sciences in Biology, UNAM, Patricia Guevara-Fefer. Doctor [email protected] Technician, UNAM, Mexico. Mexico. Professor-Researcher, in Sciences in Biology, Uni-

JULY 2016, VOL. 41 Nº 7 0378-1844/14/07/468-08 $ 3.00/0 499 ANATOMÍA, HISTOQUÍMICA Y CONTENIDO DE COMPUESTOS FENÓLICOS DE HOJAS DE Omphalea oleifera Hemsl. (EUPHORBIACEAE) EN RESPUESTA AL DAÑO POR Urania fulgens WALKER Silvia Espinosa-Matías, Roberto Enrique Llanos-Romero, Álvaro Delfino Campos Villanueva, Blanca Pérez-García, Josefina Herrera-Santoyo y Patricia Guevara-Fefer RESUMEN

Se examinaron hojas de Omphalea oleifera Hemsl. dañadas lignina y ácidos caféico, ferúlico y clorogénico fue respectiva- por acción de Urania fulgens Walker. Se describió la anato- mente 11%; 109,65; 16,58 y 0082µg·g-1 peso seco en las hojas mía foliar, se practicaron pruebas histoquímicas y se estimó dañadas, mientras que en las intactas fue de 7%; 97,65; 5,48 el contenido de lignina y ácidos fenólicos. Las características y 0,051µg·g-1 peso seco. Los resultados sugieren que el daño morfo-anatómicas fueron similares en las hojas dañadas y con- causado por la larva de Urania fulgens desencadena respues- trol, pero se observaron pequeñas diferencias histoquímicas. tas inducidas en O. oleifera que incluyen la producción y acu- Los tejidos adyacentes al daño mostraron depósitos de ligni- mulación de compuestos fenólicos y cristales de oxalato de cal- na y cristales de oxalato de calcio. El contenido estimado de cio en los tejidos de las hojas.

ANATOMIA, HISTOQUÍMICA E CONTEÚDO DE COMPOSTOS FENÓLICOS DE FOLHAS DE Omphalea oleifera Hemsl. (EUPHORBIACEAE) EM RESPOSTA DANO POR Urania fulgens WALKER Silvia Espinosa-Matías, Roberto Enrique Llanos-Romero, Álvaro Delfino Campos Villanueva, Blanca Pérez-García, Josefina Herrera-Santoyo e Patricia Guevara-Fefer RESUMO

Folhas de Omphalea oleifera Hemsl danificadas por Urania de lignina e ácidos caféico, ferúlico e clorogênico foi respecti- fulgens Walker foram examinadas. A anatomia da folha foi vamente 11%; 109,65; 16,58 e 0,082µg·g-1 peso seco nas folhas descrita, testes histoquímicos realizados e estimado o conteúdo danificadas, enquanto que, nas folhas intactas foi 7%; 97,65; de lignina e ácidos fenólicos. As características morfoanatômi- 5,48 e 0,051µg·g-1 peso seco. Os resultados sugerem que os da- cas foram similares nas folhas danificadas e no grupo contro- nos causados pela larva de U. fulgens desencadeia respostas le, entretanto foram observadas pequenas diferenças histoquí- induzidas em O. oleifera incluindo a produção e acumulação micas. Os tecidos adjacentes ao dano evidenciaram depósitos de compostos fenólicos e cristais de oxalato de cálcio nos teci- de lignina e cristais de oxalato de cálcio. O conteúdo estimado dos das folhas.

against pathogens and insects to estimate the lignin content, dehyde, acetic acid, 96% etha- me transverse sections of 25µm (Swain, 1979; El Modafar and whilst phenolic acids (caffeic, nol, water; 2:1:10:7) for 24h, thick were dehydrated through El Boustani, 2004), while caf- coumaric, ferulic, chlorogenic) then dehydrated through an an ethanol series to 100% eth- feic, p-coumaric, ferulic and were analyzed by HPLC. ethanol series until 100% etha- anol. The dehydrated material sinapic acids participate in nol and finally embedded in was dried using an CPD 030 cell wall composition, singly Materials and Methods Paraplast® blocks (Ruzin, 1999). (Bal-Tec) critical point drier, or in a range of sterified Transverse sections (8-10µm) mounted onto aluminum stubs forms (Harborne, 1988). Ano- Plant material were cut with an AO 810 rota- using double-sided carbon tape, ther defense-related adapta- tory microtome and placed on and gold coated using an Desk tions are the calcium oxalate Twenty five mature Om- slides used for the following II (Denton Vacuum) sput- crystals in any of its forms phalea oleifera leaves were histochemical tests: periodic ter-coater. Leaf sections of (Hanley et al., 2007). randomly collected before (con- acid-Schiff reagent to detect 1×1cm were similarly pro- This study is a preliminary trol) and 25 after (damaged) non soluble polysaccharides, cessed for the observations of exploration of the relationship the arrival of Urania fulgens. naphtol blue-black for proteins, the abaxial and adaxial faces. between O. oleifera and U. ful- Leaves were considered dam- oil red O for lipids, phlorogluci- gens from the plant perspective. aged when they showed signs nol-HCl for lignin, vainil- Phenolic compounds The aim was to evaluate the of the larval attack (Figure 1A, lin-HCl for hydrosoluble tannins plant responses after insect B). The sampling was done at and lugol for starch (Jensen, Lignin: Content percentage damage by comparing morpho- the Estación de Biología 1962; López et al., 2005). was estimated by triplicate, logical, anatomical and chemi- Tropical Los Tuxtlas-UNAM, Observations and micrographic using the Van Soest et al. cal features of damaged leaves Veracruz, México, located at records were made with an (1991) method. (consumed by the larvae) and 18º34’-18º36’N and 95º04’- Olympus Provis AX70 light Phenolic acids: Dried and controls. The leaves were exam- 95º09’W (García-Guzmán and microscope. ground leaves (0,1g) were ex- ined by light microscopy and Dirzo, 2001). tracted at 60ºC for 5min with scanning electron microscopy Scanning electron microscopy 5ml of 80% aqueous metha- (SEM) techniques, and its ana- Histochemistry nol. The extracts were fil- tomical and histochemical fea- Foliar morphology was ob- tered and the solvent elimi- tures described. The acid deter- Transversal sections of leaves served with a Jeol JSM5310-LV nated under reduced pressure. gent fiber procedure was used were fixed in FAA (formal- SEM. Free-hand and microto- The residue was redissolved

500 JULY 2016, VOL. 41 Nº 7 were more notorious in the periclinal wall of the epider- damaged ones (Table I). mis, giving the leaf a glandular appearance in the SEM Morphology (Figure 2K). The wall can also appear broken and the crystals O. oleifera leaves are alter- expelled (Figure 2I). Traverse nate and unifoliate. Leaf texture sections near the damaged re- is coriaceous. The lamina is gions show accumulations of complete, simple and ovate-cor- druses (Figure 3M). diform, 9-44.5cm long and 11- 39cm wide when intact. The Trichomes apex is acute to acuminate, the base is cordiform and the mar- Simple uniseriate non-glan- gin complete. Venation is re- dular trichomes are mainly ticulate, including veinlets present in the midrib and sec- (Figure 1A). The petiole apex ondary veins, and scarcely in shows two glands. The dam- the lamina of both leaf sides, aged leaves show a superficial going from one or two to five, off-white region circumscribed or absent, in a 1cm2 area. The to the edges of damaged re- cuticle of the trichomes is pa- gions (Figure 1B). When ob- pillose (Figure 3N). Big sized served in the SEM the region secretory glands (12-18) are appears as a darkened border of present in the abaxial face, 200 to 300μm wide (Figure 1C) along and parallel to the mar- that corresponds to lignin de- gin (Figure 3O). In mature posits (Figure 1D). The tissues leaves, the glands appear to of this region are morphologi- have lost their contents, leaving cally disorganized (Figure 1E). only remnants (Figure 3P). Big Leaves are amphistomatic and glands are surrounded by 5-6 Figure 1. Omphalea oleifera. A: control leaf, B: leaf damaged by larvae possess anomocytic stomata smaller glands, not visible to of the diurnal moth Urania fulgens showing ash-colored zones, C-F: with perpendicular (Figure 1F) the naked eye (Figure 3Q). SEM micrographs except C, C: the border or scar formed by lignin deposits (arrow), D: width of the scar at the edge of a damaged leaf (bra- or parallel (Figure 2G) cuticular cket), E: the damaged zone with unorganized tissues with a druse section striations surrounding the sto- Tissues (arrow), F: anomocytic stoma with perpendiculate cuticular striations ma. The cuticle can be smooth (arrow), Os: ostiole. Scale bars are 3.17mm in C; 100µm in D and E; (Figure 2H) or covered with The transverse sections of 10µm in G and F. epicuticular wax (Figure 2I). the abaxial and adaxial surfaces show a single-layered epi- Calcium oxalate dermis formed by thin walled in 5ml MeOH and filtered Results crystals (COC) cells and an evident cuticle that through a nylon membrane reacted positively to oil red O, (0.45µm pore). An aliquot of Morpho-anatomical features Damaged and control leaves indicating presence of lipids the filtrate (40µl) was ana- were similar in the damaged show druses in the epidermal (Figure 3R). The leaf is bifa- lyzed with a HPLC apparatus and control leaves; however, cells of both surfaces (Fi- cial according to the mesophyll (Merck-Hitachi LaChrom) the histochemical tests for lig- gure 2J). The druses are pro- arrangement. In transversal equipped with a RP-18 col- nin and the druse deposits jected through the external sections the adaxial face shows umn (250×4mm, 5µm particle size). The solvent system consisted of: A) methanol, TABLE I and B) KH2PO4, pH 2.4 at a MORPHOLOGICAL, ANATOMICAL AND HISTOCHEMICAL -1 flow rate of 1.5ml·min , us- CHARACTERS OF THE LEAVES OF Omphalea oleifera ing the following gradient in both samples and standards: Character Character state 15 to 55% A in 5min, 55 to Mesophyll type Bifacial 80% A in 5min, 80 to 100% Stomata disposition Amphistomatic A in 2min, 100% A in 8min Stomata type Anomocytic Cuticular ornamentation Absent or with striations parallel or perpendicular to the stoma and 100-15% A in 5min. Epidermis Single-stratified, on both leaf sides Caffeic (Sigma-Aldrich) cou- Collenchyma Lacunar maric (Sigma-Aldrich), ferulic Adaxial parenchyma Palisade, two or three cellular strata (Merck) and chlorogenic Abaxial parenchyma Spongy, three or four strata, with intercellular space (Sigma-Aldrich) acids were Lipids Present in the cuticle used as standards at 1mg·ml-1. Non-soluble polysaccharides In cell walls and laticifers Retention times were 9, 15.2, Protein bodies In cytoplasm of tissues and laticifers 13.7 and 9.2min, respectively. Lignin Present in damaged zones The samples were analysed Calcium oxalate crystals Druses distributed in epidermal cells, spongy and palisade parenchyma twice and the variation coef- and parenchyma of the vascular tissue. Mainly in damaged areas. ficient was <5%. Tannins In idioblasts

JULY 2016, VOL. 41 Nº 7 501 Figure 2. SEM micrographs. G: anomocytic stoma with parallel cuticular striations (arrowheads), H: smooth cuticle on the abaxial face of the leaf, I: epicuticular waxes on the cuticle from the adaxial face of the leaf, J: druse inside the epidermic cells from the adaxial face, K: external periclinal wall of the epidermis where druses are notorious (arrows), L: Figure 3. Light and SEM micrographs. M, druses (arrows) in the zone druses breaking the external periclinal wall of the epidermic cells (arro- damaged by the herbivore, N: non-glandular simple unicellular trichome ws). Os: ostiole, C: cuticle, Dr: druse. Scale bars are 10µm in F, G, H, over the midrib with a papillose cuticle, O: secretory gland or extrafloral K and L; 1µm in I; 5µm in J. nectary on the abaxial face, P: closer view of the extrafloral nectary (arrow), Q: the smaller secretory glands (arrows) situated around the bigger secretory gland, R: bifacial leaf with unistratified epidermis on adaxial and abaxial faces with arrowheads pointing at cuticle (note the subepidermical lacunar collen- wed accumulations of druses druses breaking the cell epidermis). Gl: gland. Scale bars are 20µm in chyma and two or three cellu- (Figure 3M). M; 10µm in N, 3,17mm in O; 1mm in P; 100µm in Q and R. lar strata of palisade parenchyma (Figure 4S). On the other Phenolic compounds hand, the abaxial face has 2002). The druses were abun- The major functions proposed three or four strata of spongy The estimated content of dant in the epidermis of dam- for COC in plants are bulk cal- parenchyma formed by cells of phenolic compounds was higher aged leaves, contrasting with cium regulation, metal detoxifi- variable size and shape and in damaged leaves (Table II), the controls, as evidenced by cation and guard against herbi- with intercellular spaces but there were no statistical the SEM observations. Calcium vores, and the increased pro- (Figure 4T). The midrib and difference between leaf groups oxalate crystals (COC) are a duction of COC has been tradi- secondary veins of damaged (Mann-Whitney U=94.5, n=15, common trait of Euphorbiaceae tionally viewed as a defense res- and control leaves characteris- p=0.4553). and can be found as styloids ponse (Finley, 1999; Jáuregui- tically feature druses in the that tear the epidermis giving Zúñiga and Moreno, 2004; Fran- parenchyma and idioblasts with Discussion the dehydrated leaf blade a ceschi and Nakata, 2005). The tannins (Figure 4U, V). Epider- rough surface, as in the genus defensive role holds up in some mal cell walls, alongside col- The lack of studies on O. Claoxylon and Micrococca (Ka- plant species (Ward et al., 1997; lenchyma, spongy and palisade oleifera remarks the importance bouw et al., 2008), as polygonal Molano-Flores, 2001; Ruiz parenchymata, reacted positive- of the foliar characters herein crystals within the mesophyll et al., 2002; Jáuregui-Zúñiga and ly to tests for the presence of observed and described. The (Levin, 1986) or as druses in Moreno, 2004; Korth et al., non-soluble polysaccharides; presence of druses that nearly the palisade parenchyma, meso- 2006; Handley et al., 2007) al- protein bodies were noticed in fill the cell lumen of the di- phyll and veins (Levin, 1986; beit not in others (Xiang and the cytoplasm content, and verse mature tissues is a char- Hussin et al., 1996; Murillo, Chen, 2004; Nagaoka et al., starch was not detected. The acter reported for the first time 2002; Kabouw et al., 2008). 2010) and some studies indicate lignin test was positive in the in the genus Omphalea, but Further studies should confirm that production of COC is in- epidermis of damaged leaves, previously observed in Conce- the consistency of this character creased even as a result of arti- appearing as a thick border in veiba guianensis (Roth, 1981) in Omphalea and determine its ficial herbivory, as reported for damaged regions (Figures 1C and species from the Conce- presence and possible variation raphides in Sida rhombifolia and 4W); the same sites sho- veibinae subtribe (Murillo, along the leaf ontogeny. (Molano-Flores, 2001).

502 JULY 2016, VOL. 41 Nº 7 control leaves, its accumulation nomic markers (Koch and in sites damaged by U. fulgens Ensikat, 2008). The striate and suggests a defense response smooth type of cuticular orna- from the plant. Although COC mentation of O. oleifera is might be present, its quantity common in Euphorbiaceae or size could have a threshold (Kulshreshtha and Ahmad, of effectiveness not met in the 1992). The striations, parallel wholly defoliated plants. or perpendicular to the stoma, Moreover, the studies about have been observed in other COC in Mexico or with Mexi- genera of Euphorbiaceae: Con- can species have focused on ceveiba Aubl., Claoxylon A. anatomy-systematics (Bárcenas- Juss., Micrococca Benth., Ery- Argüello et al., 2014, 2015 and thrococca Benth., Ricinus L., references therein), thus high- Sapium P. Browne, Alchor- lighting the Omphalea-Urania nea Sw., Acalypha L., Manihot relationship as an opportunity Miller, Jatropha L., Antidesma to expand the knowledge of the L., Bernardia Miller (Kul- COC role as an herbivory de- shreshtha and Ahmad, 1992; fense with a local species. Murillo, 2002; Kabouw et al., Non-glandular, unicellular, 2008; Cervantes et al., 2009). simple, slim and straight to The functions of cuticle orna- slightly curved trichomes featur- mentations are unclear. It has ing a papilous cuticle were ob- been proposed that they may served mainly in the leaf midrib favor colonization by fungi, and secondary veins. Similar moss or algae, which in turn trichomes have been described hinder water drainage and di- in other genera (Inamdar and minishes the photosynthetic Gangadhara, 1977; Raju and efficiency, or allow enhanced Rao, 1977; Martínez-Gordillo water drainage and light cap- Figure 4. Light and SEM micrographs. S, the lacunar collenchyma and and Espinosa-Matías, 2005, ture, particularly in plants palisade parenchyma (arrow), note the druse on the epidermal cells; T, Cervantes et al., 2009) suggest- growing under diffuse light leaf cross section showing spongy parenchyma with irregular intercellu- ing that this is a constant char- (Murillo, 2002). The latter case lar spaces (arrow); U, druses (arrows) and idioblasts with tannins (arrowheads); V, close up of idioblasts with tannins W, traverse section of a acter in the family. The reported might be occurring in O. oleif- damaged leaf with evident lignin deposits (arrows). Co: collenchyma, Pe: existence of extrafloral nectaries era, given the relatively high palisade parenchyma, Ps: spongy parenchyma, Ta: tannins, Dr: druse. in O. oleifera (Dirzo and Mota- humidity of the environment in Scale bars are 6µm in S; 38µm in T; 70 µm in U; 50µm in V and W. Bravo, 1997) was supported by which the plants grow and the the descriptions of those found absence of microorganism in the petiole (Aguirre et al., growth evidence in the SEM TABLE II 2013), and is further confirmed observations. PHENOLIC COMPOUNDS CONTENT OF by our observation of the big The leaves of O. oleifera are O. oleifera LEAV ES sized glands in the abaxial leaf bifacial and amphistomatic with Compound Damaged (µg·g-1 dry wt) Control (µg·g-1 dry wt) margin, which are similar to anomocytic stomata profusely Caffeic acid 109.65 97.65 those observed in O. diandra L. distributed in both leaf sides Coumaric acid 106.98 140.32 (Rudall, 1994b). Small glands excluding over the veins. In Ferulic acid 16.58 5.48 surrounding large glands are a Euphorbiaceae, bifacial leaves Chlorogenic acid 0.082 0.051 feature reported for the first are a constant character (Met- Lignin 11% 7% time for O. oleifera. A thorough calfe and Chalk, 1989); some analysis is needed to confirm genera have hypostomatic that extrafloral nectaries and the (Levin, 1986) or amphistomatic The invertebrate herbivores showed that an increase of small glands are a constant char- leaves (Aworinde et al., 2009) are affected by the leaf struc- COC in host oak foliage after acter in species of Omphalea. and location of the stomata is tural traits at a fine scale. It defoliation may reduce the The fine deposits of epicu- restricted to the vein in the was suggested that silica and weight of gypsy moth pupae, ticular wax in O. oleifera con- adaxial side (Kabouw et al., COC are deterrents that affect producing a high rate of mor- trast with the long filamentous 2008). Paracytic stomata are the the herbivores by the abrasion tality. This suggests that chem- structures observed in other most common in the Euphor- of its chewing mouthparts ical changes in the foliage due Euphorbiaceae (Murillo, 2002; biaceae (Raju y Rao, 1977; (Lucas et al., 2000). This neg- to natural herbivory represent Elias et al., 2008). The patterns Murillo, 2002), though aniso- ative impact was later con- an unfavorable nutritional value and morphology of wax depos- cytic and anomocytic ones are firmed (Park et al., 2009). that causes physiological weak- its have been used in plant also found (Levin, 1986; Muri- Further, an increase of calcium ness in larvae. systematics; nevertheless, simi- llo, 2002; Galeş and Toma 2006). also has been reported as a Based on previous works lar structures could result from strategy to reduce the nutri- mentioned above, our results different compounds and the ori- Phenolics tional value of leaf tissues after may suggest that the presence ginal shape can be modified by an event of herbivory damage of druses have an antiherbivore environmental factors, so cau- The damaged and control (Valentine et al., 1983). For function; further, though drus- tion should be exercised in us- leaves are statistically similar, instance, Kovacevic (1956) es are present in damaged and ing these deposits as taxo- although the content of ferulic

JULY 2016, VOL. 41 Nº 7 503 acid content is higher in the effects of damage on fitness, associated ants in tropical vege- Finley DS. (1999) Patterns of cal- damaged samples, and that of which is also related to re- tation of Los Tuxtlas, Mexico. cium oxalate crystals in young coumaric acid is higher in the source allocation patterns, Flora 208: 147-156. tropical leaves: a possible role Anderson-Prouty A, Albersheim P as an anti-herbivory defense. controls. A study on O. oleif- plant architecture, photosyn- Rev. Biol. Trop. 47: 27-31. era seedlings found that dam- thetic activity and phenological (1975) Host pathogen interactions. Plant Physiol. 56: 286-291. Franceschi VR, Nakata PA (2005) aged samples contained a patterns (Fornoni, 2011, and Calcium oxalate in plants: for- greater amount of total phenols references cited therein). Plants Aworinde DO, Nwoye DU, Jayeola AA, Olagoke AO, Ogundele AA mation and function. Annu. Rev. than healthy samples (Del Amo that are tolerant are expected (2009) Taxonomic significance Plant Biol. 56: 41-71. et al., 1986). This partial con- to have a fast growth rate in of foliar epidermis in some Galeş CR, Toma C (2006) Com- trast with our findings could relation to resource availability, members of Euphorbiaceae fa- parative anatomy of the vegeta- be explained by the sensitivity and the Omphalea-Urania as- mily in Nigeria. Res. J. Bot. tive organs of some Euphorbia and specificity of the quantifi- sociation is an interesting sub- 4: 17-28. species (Euphorbiaceae Juss.) cation method, the collection ject to study in this context. Bárcenas-Argüello ML, Terrazas T, from the Romanian Flora. season (Bernal et al., 2013), The defensive function of a Arias S (2014) Trichomes with Roman. J. Biol.-Plant Biol. 51-52: 39-47. and degree of damage (Del trait can be evidenced if the crystals in the Cephalocereus Amo et al., 1986). relative fitness of the con- Pfeiff. Areoles. Bot. Sci. 92: García-Guzmán G, Dirzo R (2001) 335-342. Patterns of leaf-pathogen infec- The content of phenolic sumed plant is increased com- tion in the understory of a compounds can rise after an pared to a plant that lacks the Barcenas-Argüello ML, Gutierrez- Castorena MCdelC, Terrazas T Mexican rain forest: incidence, insect attack (Coley, 1988; trait and grows in the same (2015) The polymorphic wedde- spatiotemporal variation and Morse et al., 1991). Plants in environment (Karban and llite crystals in three species of mechanism of infection. Am. J. general respond with mor- Baldwin, 1997). Under the Cephalocereus (Cactaceae). Bot. 88: 634-645. pho-anatomical and chemical present study settings, the exis- Micron 77: 1-8. Gillespie LJ (1997) Omphalea changes that often involve me- tence of druses and phenolics Bernal M, Llorens L, Julkunen-Tiitto (Euphorbiaceae) in Madagascar: chanical/structural barriers that are not enough to justify a de- R, Badosa J, Verdaguer D A new species and a new com- also turn difficult the contact fense response of the plant to- (2013) Altitudinal and seasonal bination. Novon 7: 127-136. or passage of opportunistic wards the herbivore, but given changes of phenolic compounds Gillespie LJ, Ambruster WS (1997) pathogens (Anderson-Prouty the reported magnitude of the in Buxus sempervirens leaves Contribution to the Guianan Flora: and cuticles. Plant Physiol. Dalechampia, Haematostemon, and Albersheim, 1975). The defoliation of O. oleifera by U. Biochem. 70: 471-482. Omphalea, Pera, Plukenetia and plants can also respond to de- fulgens (Dirzo y Mota-Bravo, Cervantes A, Terrazas T, Hernández Tragia (Euphorbiaceae) with no- foliation with an increase in 1997; Álvaro D. Campos, per- HM (2009) Foliar architecture tes on subfamily Acalyphoideae. the photosynthetic rates, differ- sonal communication), a long and anatomy of Bernardia and Smithson. Contrib. Bot. 86: 6. ential assignment of carbon, term comprehensive study, other genera of Acalyphoideae Hanley ME, Lamont BB, Fairbanks and creation of reserves of comparing populations, before, (Euphorbiaceae). Brittonia MM, Rafferty CM (2007) Plant non-structural carbohydrates, during and after the defolia- 61: 375-391. structural traits and their role in as for example in the basal tion, is necessary to character- Coley PD (1988) Effects of plant anti-herbivore defense. Perspect. growth rate and leaf lifetime on Plant Ecol. Evol. Systemat. meristems of branches; these ize the plant conditions, evalu- 8: 157-178. processes compensate the ab- ate the role of the COC and the amount and type of antiherbivore defense. Oecologia Harborne JB (1988) Introduction to sence of synthesis produced by clarify whether the observed 74: 531-536. Ecological Biochemistry. 3rd ed. the defoliation and can be des- features or other traits (second- Del Amo RS, Ramírez JG, Espejo O Academic Press. London, UK. tined to post-event growth ary metabolites, phenology, (1986) Variation of some secon- 356 pp. (Huss et al. 1996). seasonality) influence interac- dary metabolites in juvenile Huss DL, Bernardón AE, Brun JM There were no morpho-ana- tions between O. oleifera and stages of three plant species (1996) Nutrición de plantas y tomic changes between control U. fulgens, as well as deter- from tropical rain forest. J. pastizales. In Principios de ma- and damaged leaves of O. oleif- mine the biochemical, evolu- Chem. Ecol. 12: 2021-2038. nejo de praderas naturales. era. The evident response ob- tionary and ecological implica- Dirzo R, Mota-Bravo LM (1997) Oficina Regional de la FAO para served is the synthesis of lignin tions of the herbivory event. Omphalea oleifera (corcho). In América Latina y el Caribe. Santiago de Chile. 61 pp. and druses in the cells near the González-Soriano E, Dirzo R, damaged zone. Damaged lea- ACKNOWLEDGEMENTS Vogt RC (Eds.) Historia Natural Hussin KH, Wahab BA, Teh CP (1996) Comparative leaf anato- ves had, in general, a higher de los Tuxtlas, México. Uni- The authors thank Alejandro versidad Nacional Autónoma de mical studies of some Mallotus amount of the evaluated pheno- México, México. pp. 130-133. Lour. (Euphorbiaceae) species. lics and contained druses main- Martínez Mena for the micro- Bot. J. Linn. Soc. 122: 137-153. graphic records, Ana Isabel El Modafar C, El Boustani ES (2004) ly in the epidermal and paren- Inamdar JA, Gangadhara M (1977) Bieler Antolin for the image Contribución de los polifenoles a chymal cells. los mecanismos de defensa de Studies on the trichomes of Morpho-anatomical and his- layout, Fabiola Soto for the las plantas. In Regnault-Roger C, some Euphorbiaceae. Feddes tochemical studies have an im- assistance in microtechniques, Philogène BJR, Vincent C (Eds.) Repert. 88: 103-111. portant taxonomic value but and Sonia Vázquez Santana Biopesticidas de Origen Vegetal. Jáuregui-Zúñiga D, Moreno AC can also be useful to shed light and Angélica Hernández Gue- Mundi-Prensa. Madrid, España. (2004) La Biomineralización del on plant-insect interactions. rrero for their comments to the 337 pp. oxalato de calcio en plantas: Plants can recover after herbi- manuscript. Elias M, Martínez M, Espinosa- retos y potencial. Rev. Educ. Bioquím. 23: 18-23. vore attack by compensatory Matías S (2008) Caracteres folia- res del género Alchornea Sw. growth that replaces the dam- REFERENCES Jensen WA (1962) Botanical Gisto- (Euphorbiaceae) en Meso- chemistry: Principles and Prac- aged organs, a commonly stud- Aguirre A, Coates R, Cumplido- américa. Candollea 63: 39-55. tice. Freeman. San Francisco, ied response after a damaging Barragán G, Campos-Villanueva Fornoni J (2011) Ecological and evo- CA, USA. 406 pp. event that is associated to the A, Díaz-Castelazo C (2013) lutionary implications of plant Kabouw P, Van Welzen PC, Baas P, term ‘tolerance’, defined as the Morphological characterization tolerance to herbivory. Funct. Van Heuven BJ (2008) Styloid capacity to reduce the negative of extrafloral nectaries and Ecol. 25: 399-407. crystals in Claoxylon (Euphor-

504 JULY 2016, VOL. 41 Nº 7 biaceae) and allies (Claoxylinae) Kovacevic Z (1956) Food-plant se- Morse S, Wratten SD, Edward PJ, to Structure and Development. with notes on leaf anatomy. Bot. lection and the occurrence of Niemeyer MM (1991) Changes 2nd ed. Cambridge University J. Linn. Soc. 156: 445-457. plant pests (a contribution to the in the hidroxamic acid content Press. New York, USA. 110 pp. Karban R, Baldwin IT (1997) knowledge of population dy- of maize leaves with time and Ruiz N, Ward D, Saltz S (2002) Induced Responses to Herbivory. namics) (in German). Anz. after artificial damage, implica- Calcium oxalate crystals in lea- Schaedlings. 29: 97-101. tions for insect attack. Ann. University of Chicago Press, ves of Pancratium sickenberge- Appl. Biol. 119: 239-249. Chicago, IL, USA. 319 pp. Kulshreshtha K, Ahmad KJ (1992) ri: constitutive or induced de- Kite GC, Fellows LE, Lees DC, Cuticular ornamentations in Murillo J (2002) Anatomía foliar de fense? Funct. Ecol. 16: 99-105. some genera of Euphorbiaceae. la subtribu Conceveibinae (Eu- Kitchen D, Monteith GB (1991) Ruzin SE (1999) Plant Microtec- Feddes Repert. 103: 317-326. phorbiaceae). Rev. Biol. Trop. Alkaloidal glycosidase inhibitors hnique and Microscopy. Oxford 50: 9-20. in nocturnal and diurnal Uraniine Lees DC, Smith NG (1991) Food- University Press. Oxford, RU. moths and their respective food- plant associations of the Ura- Nagaoka S, Katayama H, Fujibayashi 322 pp. plant genera, Endospermum and niinae (Uraniidae) and their sys- Y, Sugimura Y (2010) Calcium Swain T (1979) Tannins and lignins. Omphalea. Biochem. Systemat. tematic, evolutionary, and ecolo- oxalate crystals in mulberry In Rosenthal GA, Cansen DH Ecol. 19: 441-445. gical significance. J. Lepidopt. leaves: No negative effect on Soc. 45: 296-347. feeding the silkworm, Bombyx (Eds.) Herbivores: Their Inte- Kite GC, Scofield AM, Lees DC, mori. J. Insect Biotechnol. raction with Secondary Plant Hughes M, Smith NG (1997) Levin GA (1986) Systematic foliar Metabolites. Academic Press. morphology of Phyllanthoideae Sericol. 79: 71-74. Alkaloidal glycosidase inhibitors London, RU. pp. 657-682. and digestive glycosidase inhibi- (Euphorbiaceae). I. Conspectus. Park S, Doege SJ, Nakata PA, tion in specialist and generalist Ann. Miss. Bot. Gard. 73: 29-85. Korth KL (2009) Medicago Valentine HT, Wallner WE, Wargo PM (1983) Nutritional changes herbivores of Omphalea diandra. López CML, Márquez-Guzmán J, truncatula-derived calcium oxa- in host foliage during and after J. Chem. Ecol. 23: 119-135. Murguía G (2005) Técnicas para late crystals have a negative impact on chewing insect per- defoliation, and their relation to Koch K, Ensikat HJ (2008) The hy- el Estudio del Desarrollo en nd formance via their physical the weight of gypsy moth pu- drophobic coatings of plant sur- Angiospermas. 2 ed. UNAM, México. properties. Entomol. Exper, pae. Oecologia 57: 298-302. faces: Epicuticular wax crystals Appl. 131: 208-215. and their morphologies, crysta- Lucas PW, Turner IM, Dominy NJ, Van Soest PJ, Robertson JB, Lewis llinity and molecular self-assem- Yamashita N (2000) Mechanical Radcliffe-Smith A (2001) Genera BA (1991) Methods for dietary bly. Micron 39: 759-772. defences to herbivory. Ann. Bot. Euphorbiacearum. Royal Botanic fiber, neutral detergent fiber and Gardens. Kew, UK. 445 pp. non-starch polysaccharides in Konno K (2001) Plant latex and 86: 913-920. relation to animal nutrition. J. other exudates as plant defense Martínez-Gordillo M, Espinosa- Raju V, Rao P (1977) Variation in Dairy Sci. 74: 3583-3597. systems: Roles of various defen- Matías S (2005) Tricomas folia- the structure and development se chemicals and proteins con- res de Croton Sección Barhamia of foliar stomata in the Ward D, Spiegel M, Saltz D (1997) tained therein. Phytochemistry (Euphorbiaceae). Acta Bot. Mex. Euphorbiaceae. Bot. J. Linn. Gazelle herbivory and interpo- 72: 1510-1530. 72: 39-51. Soc. 75: 69-97. pulation differences in calcium Roth I (1981) Structural Patterns of oxalate content of leaves of a Korth KL, Doege SJ, Park S-H, Metcalfe C, Chalk L (1989) Anatomy Tropical Barks. Borntraeger. dessert lily. J. Chem. Ecol. Goggin FL, Wang Q, Gomez of the Dicotyledon. Vol II. Berlin, Germany. 609 pp. 23: 333-346. SK, Liu G, Jia L, Nakata, PA Oxford University Press. Oxford, (2006) Medicago truncatula UK. 297 pp. Rudall P (1994a) Laticifers in Xiang H, Chen J (2004) In- mutants demonstrate the role of Molano-Flores B (2001) Herbivory and Crotonoideae (Euphorbiaceae): terspecific variation of plant plant calcium oxalate crystals as calcium concentrations effect cal- Homology and evolution. Ann. traits associated with resistance an effective defense against cium oxalate crystals formations Miss. Bot. Gard. 81: 270-282. to herbivory among four spe- chewing insects. Plant Physiol. in leaves of Sida (Malvaceae). Rudall P (1994b) Anatomy of Flo- cies of Ficus (Moraceae). Ann. 141: 188-195. Ann. Bot. 88: 387-391. wering Plants. An Introduction Bot. 94: 377-384.

JULY 2016, VOL. 41 Nº 7 505