Laticifer distribution in fig inflorescence and its potential role in the fig-fig wasp mutualism

Acta Oecologica xxx (xxxx) xxx–xxx

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Laticifer distribution in fig inflorescence and its potential role in the fig-fig wasp mutualism Cristina Ribeiro Marinhoa,∗, Rodrigo Augusto Santinelo Pereirab, Yan-Qiong Pengc, Simone Pádua Teixeiraa a

Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. do Café s/n, 14040-903 Ribeirão Preto, SP, Brazil Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes 3900, 14040-901 Ribeirão Preto, SP, Brazil c Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Menglun, Mengla, Yunnan 666303, China b

A R T I C L E I N F O

A B S T R A C T

Keywords: Evolution Ficus Flower morphology Latex Moraceae Secretory structure

Although in Moraceae the presence of laticifers is considered to be a synapomorphy, little is known about the distribution and morphology of this type of secretory structure in the reproductive organs of its species. Ficus, the largest genus of Moraceae, is characterized by an inflorescence known as syconium and by an obligate mutualistic interaction with pollinating wasps. The objectives of the present study were to evaluate the distribution and morphology of laticifers in syconia of 36 species belonging to different Ficus sections and to survey traits of taxonomic and adaptive value for the group. Syconia containing flowers in a receptive state were collected, fixed and processed for anatomical analysis. All species studied have branched laticifers distributed in the syconium receptacle, in the ostiolar bracts and in the pedicel of staminate flowers. Almost all species show laticifers in the pedicel of shorter-styled flowers. Laticifers also occur in the pedicel of longer-styled flowers in most Ficus sections, except F. curtipes (Conosycea section) and more than 75% of the studied species of the Americanae section. Laticifers are observed in the sepals of 25 of the 36 species studied and occasionally in the pistil. The presence of laticifers in the pedicel of shorter-style flowers and its absence in the pistil suggest that the distribution of this secretory structure in the fig flower was selected by pressures imposed by the fig-fig wasp mutualism. The laticifers in the pedicel of shorter-styled flowers may confer protection to the developing wasp larvae against natural enemies. However, the absence of laticifers in the pistil of most Ficus species studied was probably selected by the mutualistic relationship with the agaonid pollinating wasps since the latex could interfere with oviposition through the style, with the larval development of the pollinating fig wasps, and the emergence of pollinator offspring from their galls.

1. Introduction Laticifer is a structure specialized in the secretion of latex, an exudate composed of several substances such as polyisoprene hydrocarbons, triterpenes, sterols, fatty and aromatic acids, carotenes, phospholipids, proteins, alkaloids, inorganic compounds, etc., dispersed in a liquid with a distinct refractive index (Fahn, 1979; Ascensão, 2007). Structurally, laticifers can be nonarticulated (constituted by a single multinucleated cell which grows with the plant development) or articulated (constituted by a row of several cells whose end walls can remain intact, become porous or disappear completely). Both types can branch out or not, producing a complex system similar to tubes that permeate different tissues of the plant body (Evert, 2006). These structures act in the plant defense against herbivores, since their toxic

∗

or repellent content, which is under strong turgor pressure, is readily released to the outside in response to any injury caused to the plant (Fahn, 1979; Agrawal and Konno, 2009; Konno, 2011). In addition, the latex coagulant property acts on the sealing of wounds, preventing the entry of pathogens (Fahn, 1979; Farrell et al., 1991). In Moraceae, a family with 40 genera and about 1200 species (The Plant List, 2013), the presence of nonarticulated-branched laticifers (Metcalfe and Chalk, 1950; Fahn, 1979; Evert, 2006) is considered to be a synapomorphy (Judd et al., 2009). There are numerous studies on the distribution and morphology of laticifers in vegetative organs of Moraceae species (Tippo, 1938; Vreede, 1949; Milanez, 1954; Topper and Koek-Noorman, 1980; Davies et al., 1982; van Veenendaal and den Outer, 1990; Balaji et al., 1993; Kang et al., 2000; Jacomassi and Machado, 2003; Quintanar and Castrejón, 2004; Jacomassi et al., 2007;

Corresponding author. E-mail address: [email protected] (C.R. Marinho).

http://dx.doi.org/10.1016/j.actao.2017.10.005 Received 23 January 2017; Received in revised form 4 August 2017; Accepted 11 October 2017 1146-609X/ © 2017 Elsevier Masson SAS. All rights reserved.

Please cite this article as: Marinho, C.R., Acta Oecologica (2017), http://dx.doi.org/10.1016/j.actao.2017.10.005

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Fig. 1. Photomicrographs of longitudinal sections of Ficus inflorescences (Americanae section). A: Overview of F. citrifolia fresh syconium (image credits M.F.B. Costa). B–C: Laticifer in the syconium receptacle of F. clusiifolia (B) and F. mariae (C). D–E: Laticifer in the outer ostiolar bract of F. eximia (D) and in the inner ostiolar bract of F. pertusa (E). Staining: Toluidine blue (B–E). arrows = laticifer, br = ostiolar bracts, fl = flower, re = receptacle. Scale bars: 2 mm (A); 100 μm (B); 50 μm (C–E).

closest to the syconium receptacle, with a shorter pedicel and a longer style, form the seeds. The emergence of the wasp offspring coincides with the anthesis of the staminate flowers; thus, the female wasps leave the fig loaded with pollen and ready to restart the cycle in a new syconium (Galil and Eisikowitch, 1968a). The role of scent in the fig fig-wasp mutualism (Grison et al., 1999; Grison-Pigé et al., 2002; Dudareva and Pichersky, 2006; HossaertMcKey et al., 2010) and its associated glands (Souza et al., 2015; Machado et al., 2013) is better known than that of the latex and, consequently, of the laticifers, probably due to the higher concentration of studies on the pollination biology of fig trees (Galil and Eisikowitch, 1968a; Anstett et al., 1997; Cook and Rasplus, 2003) than on the commensal or parasitic microfauna of the mutualism (Cruaud et al., 2011). Considering the ecological role of Ficus syconium and the scarcity of information about the protective glands in these inflorescences, we compared here the distribution and morphology of laticifers in syconia of 21 Ficus species belonging to the Americanae section and 15 other species belonging to nine additional sections, namely Conosycea, Dammaropsis, Ficus, Galoglychia, Pharmacosycea, Sycidium, Sycocarpus, Sycomorus and Urostigma. Traits of taxonomic and adaptive value were surveyed for the group. Evolutionary aspects were also addressed using the phylogeny published by Cruaud et al. (2012) as reference.

Palhares et al., 2007; Ramadan et al., 2008; Duarte et al., 2012; Alonso et al., 2013; Araújo et al., 2014; Bercu and Popoviciu, 2014; Kajii et al., 2014; Sharma et al., 2014); however, studies in reproductive organs are scarce. In organs related to plant reproduction, studies of laticifers have been reported for Sorocea bonplandii (Souza and Rosa, 2005), Brosimum gaudichaudii (Jacomassi et al., 2010), Maclura tinctoria (Oyama and Souza, 2011), and some Ficus species (Machado et al., 2013; Subramanian et al., 2013; Souza et al., 2015). In Ficus, 11 out of the approx. 750 described species had their laticifers studied in reproductive organs. Besides being the richest genus of Moraceae, Ficus exhibits a wide diversity of habits and geographic distribution, as well as a unique type of inflorescence called syconium (Clement and Weiblen, 2009; Judd et al., 2009). The syconium exhibits an urn-shaped receptacle containing several diclinous flowers, and opens to the outside through an ostiole, which is lined by bracts (Datwyler and Weiblen, 2004; see Fig. 1A). It is through the ostiole that the fig wasps enter the syconium, pollinate the flowers and lay eggs in some of them, ensuring the production of seeds and wasp offspring and setting up a very specialized and one of the most currently studied mutualistic interactions (Galil and Eisikowitch, 1968a; Anstett et al., 1997; Cook and Rasplus, 2003). Female pollinating wasps are attracted to receptive syconia (B phase) by a scent produced in osmophores present on the surface of the syconium and on the ostiolar bracts (Souza et al., 2015). After entering the syconium the wasps oviposit while they pollinate the pistillate flowers. The oviposition generally occurs in flowers with ovaries located closer to the fig cavity and therefore with longer pedicels and shorter styles. In this process, the pollinating wasp introduces its ovipositor through the style and deposit an egg between the inner integument and the nucellus. The flowers with the ovary

2. Material and methods Syconia at the receptive phase (B phase) of 36 species of Ficus (Table 1) were collected and fixed in FAA50 (Johansen, 1940) or neutral buffered formalin (Lillie, 1965). Vouchers were deposited in the HITBC, 2

Monoecious

Americanae Americanae Americanae Americanae Americanae Americanae Americanae

Ficus boliviana C.C.Berg

calyptroceras (Miq.) Miq. carautana L.J.Neves & Emygdio citrifolia Mill. clusiifolia Schott costaricana (Liebm.) Miq.

crocata (Miq.) Mart. ex Miq. cyclophylla (Miq.) Miq. eximia Schott gomelleira Kunth & C.D.Bouché

Ficus Ficus Ficus Ficus Ficus

Ficus Ficus Ficus Ficus

3

Americanae Americanae Americanae Americanae Americanae Americanae Conosycea Conosycea Dammaropsis Ficus Galoglychia Pharmacosycea Sycidium Sycidium

Ficus luschnathiana (Miq.) Miq. Ficus mariae C.C.Berg, Emygdio & Carauta

Ficus obtusifolia Kunth Ficus pertusa L.f. Ficus roraimensis C.C.Berg

Ficus trigona L.f.

Ficus curtipes Corner

Ficus microcarpa L.f. Ficus pseudopalma Blanco

Ficus Ficus Ficus Ficus Ficus

Sycomorus Sycomorus Sycomorus Sycomorus

Ficus Ficus Ficus Ficus

Ficus religiosa L.

Urostigma

Sycocarpus

Ficus septica Burm.f.

auriculata Lour. racemosa L. tikoua Bureau variegata Blume

Sycocarpus

Ficus hispida L.f.

pumila L. lyrata Warb. obtusiuscula (Miq.) Miq. montana Burm.f. ulmifolia Lam.

Americanae Americanae

Ficus holosericea Schott Ficus lauretana Vázq.Avila

Americanae Americanae Americanae Americanae

Monoecious

Americanae

Monoecious

Gynodioecious/male and female Gynodioecious/male and female Gynodioecious/female Monoecious Gynodioecious/female Gynodioecious/female

Gynodioecious/male Monoecious Monoecious Gynodioecious/male Gynodioecious/female

Monoecious Gynodioecious/male

Monoecious

Monoecious

Monoecious Monoecious Monoecious

Monoecious Monoecious

Monoecious Monoecious

Monoecious Monoecious Monoecious Monoecious

Monoecious Monoecious Monoecious Monoecious Monoecious

Monoecious

Monoecious

Americanae

Ficus americana subsp. guianensis (Desv. ex Ham.) C.C.Berg ´mathewsii form' = Ficus guianensis Desv. ex Ham. Ficus americana subsp. guianensis (Desv. ex Ham.) C.C.Berg ´parkeriana form' = Ficus parkeriana (Miq.) Sandwith Ficus aurea Nutt.

Sexual expression/Studied syconium

Section

Species

Table 1 Information of Ficus species used in this study. Species are ranked by section.

Xishuangbanna Tropical Botanical Garden - China Ribeirão Preto - Brazil University of the Philippines Diliman - Philippines Ribeirão Preto - Brazil Ribeirão Preto - Brazil Ribeirão Preto - Brazil Ribeirão Preto - Brazil University of the Philippines Diliman - Philippines Xishuangbanna Tropical Botanical Garden - China University of the Philippines Diliman - Philippines Ribeirão Preto - Brazil Rio de Janeiro - Brazil Ribeirão Preto - Brazil University of the Philippines Diliman - Philippines Xishuangbanna Tropical Botanical Garden - China

Rio de Janeiro - Brazil

Galia - Brazil Ribeirão Preto - Brazil Manaus - Brazil

Picinguaba - Brazil Sooretama - Brazil

Santa Teresa - Brazil Sena Madureira - Brazil

Cuiabá - Brazil Rio de Janeiro - Brazil Ribeirão Preto - Brazil Picinguaba - Brazil

Corumbá - Brazil Corumbá - Brazil Ribeirão Preto - Brazil Rio de Janeiro - Brazil Guápiles - Costa Rica

Sena Madureira - Brazil

Ojochal - Costa Rica

Manaus - Brazil

Manaus - Brazil

Collect sites

Oriental

Oriental Australasian/Oriental Oriental Oriental

Oriental

Oriental

Oriental Afrotropical Neotropical Oriental Oriental

Oriental Oriental

Oriental

Neotropical

Neotropical Neotropical Neotropical

Neotropical Neotropical

Neotropical Neotropical

Neotropical Neotropical Neotropical Neotropical

Neotropical Neotropical Neotropical Neotropical Neotropical

Neotropical

Neotropical

Neotropical

Neotropical

Native distribution

HITBC 58160

SPFR/S.P.Teixeira & C.D.Souza 78 RB 422367 SPFR/S.P.Teixeira et al. 84 PUH 1653

PUH 10

HITBC 23038

SPFR/S.P.Teixeira et al. 83 SPFR/S.P.Teixeira et al. 80 SPFR/S.P.Teixeira et al. 82 SPFR/S.P.Teixeira & C.D.Souza 85 PUH 1649

SPFR/S.P.Teixeira et al. 81 PUH 1645

spirit collection at Botany Lab of the USP spirit collection at Botany Lab of the USP SPFR/R.A.S.Pereira et al. 179 SPFR/R.A.S.Pereira et al. 178 SPFR/S.P.Teixeira et al. 79 RB 342079 spirit collection at Botany Lab of the USP SPFR/P.C.Costa & F.H.A.Farache 60 RB 224477 SPFR/R.A.S.Pereira et al. 138 spirit collection at Botany Lab of the USP SPFR/A.P.Fontana 7839 spirit collection at Botany Lab of the USP SPFR/R.A.S.Pereira et al. 124 SPFR/G.S.Siqueira 819, SPFR/P.C.Costa & M.F.B. Costa 47 SPFR/R.A.S.Pereira et al. 158 SPFR/R.A.S.Pereira et al. 127 spirit collection at Botany Lab of the USP spirit collection at Botany Lab of the USP HITBC 22860, HITBC 64145

SPFR/A.C.C.Rezende 20

SPFR/A.C.C.Rezende 1

FCFRP/

FCFRP/

FCFRP/

FCFRP/

FCFRP/

FCFRP/

FCFRP/

Herbarium/Voucher or collector number

C.R. Marinho et al.

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Table 2 Distribution of laticifers in inflorescences and flowers of Ficus species. FSS = flor with shorter style; FLS = flower with longer style; ? = unobserved material. Species are ranked by section. Ficus species

Section

Syconium receptacle

Ostiolar bracts

Sepals

Pistillate flower Pedicel

Ficus americana subsp. guianensis ´mathewsii form' Ficus americana subsp. guianensis ´parkeriana form' F. aurea F. boliviana F. calyptroceras F. carautana F. citrifolia F. clusiifolia F. costaricana F. crocata F. cyclophylla F. eximia F. gomelleira F. holosericea F. lauretana F. luschnathiana F. mariae F. obtusifolia F. pertusa F. roraimensis F. trigona F. curtipes F. microcarpa F. pseudopalma F. pumila F. lyrata F. obtusiuscula F. montana F. ulmifolia F. hispida F. septica F. auriculata F. racemosa F. tikoua F. variegata F. religiosa

Americanae Americanae Americanae Americanae Americanae Americanae Americanae Americanae Americanae Americanae Americanae Americanae Americanae Americanae Americanae Americanae Americanae Americanae Americanae Americanae Americanae Conosycea Conosycea Dammaropsis Ficus Galoglychia Pharmacosycea Sycidium Sycidium Sycocarpus Sycocarpus Sycomorus Sycomorus Sycomorus Sycomorus Urostigma

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + + + + + + ? + + + + + + + ? + + ? + + +

+ – + + + + – + + + – + + + – + + + – + – + + – + (staminate) + + + + – + – – + – +

FSS

FLS

+ + + + + + + + + + + + + + + + + + + + + + + – – + + + ? + + ? + ? ? +

– – – – – – – – – – + – – – – + + – – + – – + ? ? + + ? + + + + + + + +

Staminate flower Pistil

Pedicel

Stamen

– – – – – – – – – – – – – – – – – – – – – – – – – – + – – + + – – – – +

+ + + ? + + + ? + + + + + + ? ? + + + + + + + – + ? + ? ? ? + ? + + ? +

– – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –

species studied, laticifers are observed in the pedicel of all pistillate flowers, except F. curtipes (Conosycea section) where it is absent in the pedicel of longer-styled flowers. In the gynodioecious species of the Sycidium and Sycocarpus sections in which syconia of both male and female trees were studied, laticifers are present in the pedicel of both shorter- and longer-styled flowers. In gynodioecious species of the Sycomorus section we studied only syconia of female trees and laticifers were observed in the pedicel of longer-styled flowers (Table 2). Laticifers are generally absent in the pistil, occurring only in the ovary wall of F. hispida and in the ovary and style of F. septica, F. religiosa and F. obtusiuscula. Laticifers are also absent in the stamens (Table 2). However, they are found in the sepals (Fig. 2E) of pistillate and staminate flowers of 25 of the 36 species studied. The variation in laticifer distribution in the syconium of Ficus species associated with the latest phylogeny of the group (Fig. 3) indicates that there was a loss of laticifers in the sepals, in the pedicel of the pistillate flowers with a longer or shorter style and in the pistil of some species. The laticifers in the pedicel of shorter-styled flowers were lost at least two times in Ficus (i.e., sections Ficus and Dammaropsis). In the pedicel of longer-styled flowers, the absence of laticifers seems to be restricted to some species of the subgenus Urostigma, sections Americanae and Conosycea (Fig. 3). There was a multiple independent loss of laticifers in sepals, apparently restricted to section Americanae and subgenus Sycomorus (i.e., sections Dammaropsis, Sycocarpus and Sycomorus). Although laticifers are absent in the pistil of most species studied, they occurred independently in representatives of sections

PUH, RB and SPFR herbaria (Table 1). The collected material was dehydrated in ethanol series, embedded in methacrylate (Historesin - Leica) and cut into 2–5 μm thick crosswise and lengthwise sections in a rotary microtome. The obtained sections were stained with 0.05% Toluidine blue (O'Brien et al., 1964) and assembled under a coverslip with synthetic resin. The slides were analyzed with a Leica DME photonic microscope and photomicrographs were obtained with a Leica DM 4500 B light microscope coupled to a Leica DFC 320 digital camera. The evolutionary aspects of laticifer distribution in the syconia of Ficus were assessed by plotting these data according to the latest Ficus phylogeny (Cruaud et al., 2012). 3. Results All species studied show laticifers of the branched type with thin cell walls immersed in the syconia tissues. These structures occur in the receptacle, in the outer and inner ostiolar bracts and in the pedicel of staminate flowers, immersed in the parenchyma, associated or not with the vascular bundles (Table 2, Figs. 1–2). In pistillate flowers, laticifers are present in the pedicel of shorterstyled flowers of almost all species, except for male trees of F. pseudopalma and F. pumila. In the Americanae section laticifers occur in the pedicel of all shorter-styled flowers and in the pedicel of longer-styled flowers of four of the 21 species studied (Fig. 2A–C): Ficus crocata, F. luschnathiana, F. mariae and F. roraimensis. In the other monoecious 4

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Fig. 2. Photomicrographs of longitudinal sections of Ficus inflorescences (Americanae section). A–B: Laticifers in the pedicel of the flower with short style of Ficus americana subsp. guianensis ‘mathewsii form’ (A) and F. clusiifolia (B). C: Laticifer in the pedicel of the flower with long style of F. roraimensis. D: Laticifer in the pedicel of the staminate flower of F. cyclophylla. E: Laticifer in F. clusiifolia sepal. Staining: Toluidine blue. arrows = laticifer. Scale bars: 200 μm (A); 100 μm (B–D); 50 μm (E).

the style, the ovipositor would be coated with it, which probably interferes with the ovipositor sensilla (Ghara et al., 2011) and, consequently, with the oviposition process. Laticifers are virtually present in the pedicel of shorter-styled flowers of all species studied (except F. pseudopalma and F. pumila), suggesting that they can have been selected by the pressure exerted by non-pollinating fig wasps that oviposit from the external surface of the fig. It is interesting to note that F. pseudopalma and F. pumila seem to have low (or no) pressure of non-pollinating fig wasps and do not present these protective structures in the pedicel of their shorter-styled flowers. Only one species of Philotrypesis is associated with F. pseudopalma (L.J.V. Rodriguez, unpublished data) and no non-pollinating fig wasps have been recorded for F. pumila (Yokoyama and Iwatsuki, 1998; Bain et al., 2015). Ficus pseudopalma and F. pumila belong to different lineages of Ficus (i.e., sections Dammaropsis and Ficus, respectively) indicating that the laticifers in the pedicels of shorter-styled flowers were lost at least two times independently. However, the large number of wasp species that oviposit through the fig wall (Borges, 2015), including some Apocrypta and Philotrypesis that oviposit through the flower pedicels as well (Compton and Nefdt, 1988; Elias et al., 2012), indicates that non-pollinating fig wasps overcame the presence of laticifers in the fig receptacle and flower pedicels. The occurrence of laticifers in the pedicel of flowers with shorter-

Pharmacosycea, Sycocarpus and Urostigma (Fig. 3). 4. Discussion Our results showed that laticifers seem to be ubiquitously present in the syconium receptacle, ostiolar bracts and pedicel of staminate flowers, but absent in the stamen of all species studied. On the contrary, variation in laticifer distribution in pistillate flowers seems to have no relation with the sexual expression or phylogenetic relationship of the species studied, but rather with the mutualistic interaction with the pollinating wasps since laticifers are virtually absent in organs with which the pollinating wasps interact (i.e., pistil and stamen). Indeed, Ficus latex presents various repellent compounds such as rubber, terpenes, alkaloids, proteases and peroxidases (Kang et al., 2000; Perelló et al., 2000; Agrawal and Konno, 2009; Kumari et al., 2010; Konno, 2011; Lazreg-Aref et al., 2012; Araújo et al., 2014; Mnif et al., 2015; Souza et al., 2015) that could disturb the larval development of the fig wasp or the process of pollen presentation (e.g., female and male fig wasps in many fig tree species manipulate or even cut the anthers to expose pollen grains, Galil and Eisikowitch, 1968b; Galil and Eisikowitch, 1974; Michaloud et al., 2005). Additionally, during the oviposition process the wasp ovipositor damages the style tissue when it is introduced (Jansen-González et al., 2012). So, if there was latex in 5

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Fig. 3. Phylogenetic hypothesis modified from Cruaud et al. (2012) with the terminal branches colored showing the presence of laticifers in the pedicel of flowers with short (FSS, blue squares) and long style (FLS, red squares), in the sepals (green squares) and in the pistil (yellow squares) of the studied species. ? = not analyzed. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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styled flowers is almost ubiquitous in Ficus, suggesting their biological importance for the fig-fig wasp mutualism. However, laticifers are absent from the pedicel of longer-styled flowers in most of the studied species of the Americanae section and in F. curtipes (Conosycea section). The selective pressures that drove the occurrence of laticifers in the pedicels of longer-styled flowers probably differed in gynodioecious and monoecious Ficus species due to the spatial arrangement of flower ovaries inside the syconia. In gynodioecious species, ovaries are arranged in a single layer in both female and male inflorescences. Conversely, in monoecious species ovaries are arranged in multiple layers, with the pollinating wasps preferentially ovipositing in the shorter-styled flowers and seeds being produced in the longer-styled flowers (Anstett et al., 1996; Nefdt and Compton, 1996). Therefore, the single-layer flower arrangement might explain the apparently omnipresence of laticifers in the pedicels of longer-styled flowers in gynodioecious Ficus species and in F. racemosa (in which the monoecy reverted from the gynodioecy condition, Cruaud et al., 2012) since all pistillate flowers in a female fig would be equally under pressure against herbivory. In the monoecious species, the question remains open to investigation, but the variation in distribution, especially in the Americanae section, is possibly related to the more complex spatial structure of the ovaries, that can lead to particular selective pressures according to the species characteristics. For example, ovary gallers are represented by internally (e.g., the pollinators and some non-pollinators, Galil et al., 1970; Jousselin et al., 2001) and externally ovipositing species (Cruaud et al., 2011) that compete for the ovaries of shorterstyled flowers. Experimental data, however, have shown that ovary gallers tend to use longer-styled flowers in situations of intense site competition (Anstett et al., 1996; Nefdt and Compton, 1996). Therefore, the herbivory pressure upon longer-styled flowers in monoecious fig tree species is probably associated with ecological factors (i.e., wasp and tree population densities) as well as the syconium morphology (i.e., number of flowers and their arrangement). Indeed, syconium morphology of the Americanae species studied is quite variable, ranging from 4 to 20 mm in size at the B phase (data not shown). Laticifers in sepals were lost several times within Ficus since unrelated lineages in the phylogeny do not present laticifers in this organ. This phylogenetic pattern is more likely due to physical restriction of the sepals (i.e., small and thin) than to ecological factors. In fact, no ecological/biological role related to the obligate mutualism is known for the fig sepals. Conversely, among the Moraceae members with a perianth, Ficus species are those that present the less evident sepals. Other species of the family, such as Artocarpus heterophyllus, Castilla elastica, Dorstenia cayapia, Maclura tinctoria and Sorocea bonplandii, however, have more developed sepals with laticifers ever present (C.R. Marinho, unpublished data). The absence of laticifers in the pistil of most Ficus species studied was probably selected by the mutualistic relationship with the agaonid pollinating wasps. As latex of fig trees has repellent compounds (Agrawal and Konno, 2009; Konno, 2011), it could interfere with (1) oviposition (which is done through the style), (2) larval development of the pollinating fig wasps (Jansen-González et al., 2012), and (3) the emergence of pollinator offspring from their galls. Indeed, the presence of laticifers in the pistil of other genera of Moraceae (C.R. Marinho, unpublished data) supports the hypothesis that pistil laticifers were lost in several Ficus species. The distribution of laticifers probably presents a biological and unifying value for Ficus, as other genera of Moraceae belonging to different tribes present a distinct pattern of laticifer distribution in the flowers, including Castilleae that is the sister group of Ficus. In Artocarpus heterophyllus (Artocarpeae), Brosimum gaudichaudii, Dorstenia cayapia (Dorstenieae), Castilla elastica (Castilleae), Maclura tinctoria (Maclureae) and Sorocea bonplandii (Moreae), for instance, laticifers are found in the ovary and style, while in B. gaudichaudii, M. tinctoria and S. bonplandii, laticifers also occur in the filaments (C.R. Marinho, unpublished data). It is noteworthy that the fig flowers are enclosed

within the syconium (Datwyler and Weiblen, 2004), so a stronger selective pressure is expected for the occurrence of protective secretory structures in more exposed inflorescence parts, such as the receptacle, ostiolar bracts and flower pedicels. Considering that the flowers of other Moraceae genera present other types of inflorescence in which their flowers are exposed (Clement and Weiblen, 2009), our results also support the protective role explanation for presence of latex in the flowers of these species. Besides, supposing that the inflorescence closure relaxed the selective pressure for chemical defenses in the fig flowers, we can hypothesize that the appearance of the urn-shaped inflorescence was a key evolutionary event for the establishment of the fig-fig wasp mutualism. 5. Conclusions Laticifers seem to play an important role in fig trees and their pollinating wasps, as their distribution in the syconium potentially favors the mutualism: laticifers are virtually absent in flower organs with which pollinators interact (i.e., pistil and stamen), but they are generally present in more exposed structures that protect both seeds and pollinating larvae against natural enemies. Plant secretory structures present in flowers/inflorescences are generally associated with animal attraction (e.g., osmophores, nectaries, elaiophores and resin glands), as they directly or indirectly act as a reward, especially in the form of food (Teixeira et al., 2014). However, secretory structures involved in plant defense being also used for protection of animals are less frequent in nature. In this context, the present study opens new questions to be investigated in the fig-fig wasp mutualism. Author contribution C.R. Marinho and S.P. Teixeira conceived and designed the experiments. C.R. Marinho performed the experiments. All the authors analyzed the data. C.R. Marinho, R.A.S. Pereira and S.P. Teixeira wrote the manuscript. Y.Q. Peng edited the text. Acknowledgments We thank Camila D. Souza, Marina F. B. Costa, Luciano Palmieri (University of São Paulo, Ribeirão Preto, Brazil), and Lillian J. V. Rodriguez (University of the Philippines Diliman) for the Ficus samples. We also thank Elettra Greene for revising the English. This work was supported by Fapesp [grant numbers 2013/21794-5 and 2014/074533]; and Cnpq [grant numbers 306078/2014-7 and 303493/2015-1]. References Agrawal, A.A., Konno, K., 2009. Latex: a model for understanding mechanisms, ecology, and evolution of plant defense against herbivory. Annu. Rev. Ecol. Evol. Syst. 40, 311–331. https://dx.doi.org/10.1146/annurev.ecolsys.110308.120307. Alonso, A.A., Mendonça, M.S., Reis, R.S., Biondo, P.L.T.A., Alonso, R.R.P., 2013. Laticifers distribution in secondary phloem of the Amazon wood species. Com. Sci. 4, 212–215. Anstett, M.C., Bronstein, J.L., Hossaert-McKey, M., 1996. Resource allocation: a conflict in the fig/fig wasp mutualism? J. Evol. Biol. 9, 417–428. https://dx.doi.org/10.1046/ j.1420-9101.1996.9040417.x. Anstett, M.C., Hossaert-McKey, M., Kjellberg, F., 1997. Figs and fig pollinators: evolutionary conflicts in a coevolved mutualism. Tree 12, 94–99. https://dx.doi.org/10. 1016/S0169-5347(96)10064-1. Araújo, N.D., Coelho, V.P.M., Ventrella, M.C., Agra, M.F., 2014. Leaf anatomy and histochemistry of three species of Ficus sect. Americanae supported by light and electron microscopy. Microsc. Microanal. 20, 296–304. https://dx.doi.org/10.1017/ S1431927613013743. Ascensão, L., 2007. Estruturas secretoras em plantas. Uma abordagem morfo-anatómica. In: Figueiredo, A.C., Barroso, J.G., Pedro, L.G. (Eds.), Potencialidades e aplicações das plantas aromáticas e medicinais. Curso teórico-prático, 3a ed. Faculdade da Universidade de Lisboa – Centro de Biotecnologia Vegetal, Lisboa, pp. 19–28. Bain, A., Tzeng, H., Wu, W., Chou, L., 2015. Ficus (Moraceae) and fig wasps (Hymenoptera: Chalcidoidea) in Taiwan. Bot. Stud. 56, 1–12. https://dx.doi.org/10. 1186/s40529-015-0090-x. Balaji, K., Subramanian, R.B., Inamdar, J.A., 1993. Occurrence of non-articulated laticifers in Streblus asper Lour. (Moraceae). Phytomorphol 43, 235–238.

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