Botany

Comparative anatomy of the fig wall (, )

Journal: Botany

Manuscript ID cjb-2018-0192.R2

Manuscript Type: Article

Date Submitted by the 12-Mar-2019 Author:

Complete List of Authors: Fan, Kang-Yu; National Taiwan University, Institute of Ecology and Evolutionary Biology Bain, Anthony; national Sun yat-sen university, Department of biological sciences; National Taiwan University, Institute of Ecology and Evolutionary Biology Tzeng, Hsy-Yu; National Chung Hsing University, Department of Forestry Chiang, Yun-Peng;Draft National Taiwan University, Institute of Ecology and Evolutionary Biology Chou, Lien-Siang; National Taiwan University, Institute of Ecology and Evolutionary Biology Kuo-Huang, Ling-Long; National Taiwan University, Institute of Ecology and Evolutionary Biology

Keyword: Comparative Anatomy, Ficus, Histology, Inflorescence

Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? :

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Comparative anatomy of the fig wall (Ficus, Moraceae)

Kang-Yu Fana, Anthony Baina,b *, Hsy-Yu Tzengc, Yun-Peng Chianga, Lien-Siang

Choua, Ling-Long Kuo-Huanga

a Institute of Ecology and Evolutionary Biology, College of Life Sciences, National

Taiwan University, 1, Sec. 4, Roosevelt Road, Taipei, 10617, Taiwan

b current address: Department of Biological Sciences, National Sun Yat-sen University,

70 Lien-Hai road, Kaohsiung, Taiwan.Draft

c Department of Forestry, National Chung Hsing University, 145 Xingda Rd., South

Dist., Taichung, 402, Taiwan.

* Corresponding author: [email protected]; Tel: +886-75252000-3617;

Fax: +886-75253609.

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Abstract

The genus Ficus is unique by its closed inflorescence (fig) holding all flowers inside its cavity, which is isolated from the outside world by a fleshy barrier: the fig wall. The fig wall is the main structure of the fig giving its shape but the wall has also important ecological functions such as protection of fig seeds and larvae. Nevertheless, the fig wall anatomy is poorly understood. This study aims to examine the fig wall anatomy of 22 Ficus taxa (21 , one species having two varieties) in Taiwan in order to reveal the diversity in anatomy of the fig Draftwall. We found that these 21 fig species exhibited a great variety in fig wall anatomy, from the simplest parenchymatic wall to complex fig walls. Fig walls of 12 sampled taxa developed aerenchyma and sclerenchyma formations whereas seven taxa had fig walls containing tanniferous cells. Five anatomical types of fig walls have been identified according to the presence or absence of the different differentiated tissues. These types are distributed among the Ficus subgenera. Further studies on tissue differentiations of the fig wall should be investigated in other Ficus species as well as the ecological functions of the fig wall.

Keywords: Comparative Anatomy; Ficus; Histology; Inflorescence

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1. Introduction

The Moraceae family comprises about 38 genera and 1180 species (Christenhusz and

Byng 2016) displaying a great diversity of life history traits with few synapomorphic

characters such as laticifers, anatropous ovules, and apical placentation (Sytsma et al.

2002). Among the six Moraceae tribes (Clement and Weiblen 2009), the Ficeae tribe is

monogeneric with the single genus Ficus (Berg 2001) defined by a common feature: a

closed inflorescence with a single tight orifice (ostiole) closed by bracts called fig (or

syconium) (Berg and Corner 2005). Draft The Moraceae phylogeny shows that the Ficeae tribe is not basal and the

inflorescence morphology within the Moraceae family has evolved in many directions

(Clement and Weiblen 2009). This diversity is also present within the tribes. For instance,

figs display many shapes from the ellipsoid figs of Ficus dammaropsis measuring about

10cm in diameter with conspicuous lateral bracts to the globose minute figs of F.

caulocarpa measuring half a centimetre (Berg and Corner 2005) or to the Australian

banana fig, F. pleurocarpa (Dixon 2003). In addition to the fig morphology, Ficus species

are mostly famous for being pollinated by minute wasps from the Agaonidae family

(: Chalcidoidea) and solely by the wasps of this family. Together, the fig

tree and the pollinating wasp are mutualistic partners: the fig provides oviposition sites

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for the agaonid wasp which bring pollen inside the fig in order to pollinate the enclosed flowers (Kjellberg et al. 2005). This nursery pollination mutualism is targeted by many nonpollinating wasp (NPFW) species parasitizing either the fig ovules or fig wasp larvae

(Bronstein 1991; Tzeng et al. 2008). These NPFWs can drastically reduce the number of seeds and pollinating wasps produced in a single fig (Cardona et al. 2013). The specificity of these NPFWs is their way to reach the fig ovules or the fig wasp larvae inside the figs.

Indeed, they are laying eggs from outside the figs, using their long ovipositor (Ghara et al. 2011). They are not using the fig ostiole to penetrate the fig but pass through the fig Draft wall.The fig wall is the tissue between the ovules and the outside world (Fig. 1). The thickness of the fig wall increases during the fig development with the size of the fig

(Galil et al. 1970). Moreover, some figs of Ficus erecta var. beecheyana, in Taiwan, have a fig wall thick enough to protect their flowers from nonpollinating fig wasp oviposition

(Tzeng et al. 2014). Other than laticifers (Marinho et al. 2017), the anatomy of the fig wall is poorly understood. To our knowledge, the general description is “the fig wall generally parenchymatic and contains some 30-40 cell layers. Various sclerified cell layers may occur […] and contains many laticifers, tannin cells, and a vascularization”

(Verkerke 1989). This general description based on information from different reviewed studies but the fig wall can contain more cell layers than in the Verkerke’s description reaching up to 48 cell layers in F. ingens in South (Baijnath and Naicker 1989).

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One of the common feature seems to be that, when it occurs, sclerenchyma becomes a

larger part of the fig wall during the fig development (Galil et al. 1970; Verkerke 1986;

Baijnath and Naicker 1989) but not all the fig species have a hardened fig wall (Verkerke

1988). This statement leads to the question why some fig species have developed a

hardened fig wall. Considering the cost of the NPFWs (Cardona et al. 2013) and the

adaptation of these NPFWs to the oviposition through the fig wall (Ghara et al. 2011), the

fig wall may be an important organ for the fig trees to manage the parasitism by NPFWs.

For instance, some closely related fig species, such as F. caulocarpa and F. subpisocarpa, Draft have a very a different NPFW community (Bain et al. 2015): 20 species for F.

subpisocarpa and only two species for F. caulocarpa. Then, is the fig wall of these two

species very different? Another example is F. erecta var. beecheyana which can have a

very thick fig wall excluding any NPFWs. What does the fig wall of F. erecta var.

beecheyana consist of? And more generally, what are the fig walls made of? Other than

the simple description from Verkeke’s work (1988), we have little information except

that fig walls display some anatomical diversity. Moreover, what happens in dioecious

fig species? In these species, the male trees bear figs with pollinating fig wasps and are

the targets of many NPFWs whereas the female trees are producing only seeds and

NPFWs are rarely breeding in female figs (Wu et al. 2013). If the fig wall is ecologically

linked to the presence of the NPFWs, we can expect to see morphological differences

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between male and female trees.

Thus, this study aims to investigate the fig wall anatomy of most of the Taiwan Ficus species (21 species, one of which has two varieties, from six subgenera) and tentatively link the anatomical structures to ecological functions such as the parasitism from NPFWs.

We also discuss the effect of phylogeny on the distribution of the anatomical structures within the genus Ficus.

2. Methods Draft 2.1. Fig development and reproductive biology of pollinating fig wasps

The pollinating fig wasps grow as larva inside the figs and mate shortly after the eclosion of their gall (pupal case) when they are still inside their natal fig (Kjellberg et al. 2005).

Only the winged female pollinating fig wasps will leave the fig to find another fig of the same species because pollinating fig wasps pollinate only one fig tree species (few exceptions to this rule have been found (Compton et al. 2009)). Once the female pollinating wasp has found a receptive fig (see below for the descriptions of fig developmental phases), it enters inside the fig to pollinate it and lay eggs. In the figs, pollinating and nonpollinating fig wasps have a very similar life cycle but, instead of entering to figs to lay eggs, NPFWs lay eggs from outside of the fig and have to penetrate

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the fig wall with their ovipositors (Weiblen et al. 2002).

Based on the definition by Galil and Eisikowitch (1968), fig development is divided

into five phases. The A phase (prefemale phase) is the earliest stage, in which all the

flowers are immature. In the B phase (female phase), the female flowers are receptive,

and pollinating fig wasps can pollinate them, thus entering the figs through the ostiole.

During the C phase (interfloral phase), both the fig wasp larvae and seeds develop. The

D phase (male phase) begins when the first male wasp hatches. Subsequently, the wasps

mate, and the males dig an exit for the female winged wasps, which leave the fig to find Draft another receptive fig. During the E phase (post-floral phase), the figs ripen and attract

frugivores to disperse the seeds.

During their larval stage, NPFWs exhibit a great diversity of hosts and diets: for

instance, some larvae feed on the tissue growing from a gall whereas other NPFW

species feed on the larva of another fig wasp species (they are parasitoid). Most NPFWs

do not feed as adult. Thus, depending on their larval diet, NPFWs will appear on figs to

lay their eggs during two of the developmental phases: During the late B phase or early

C phase, if the NPFW species are gall makers; and later during the C phase if they are

inquiline (gall thief) or parasitoid (Kerdelhué and Rasplus 1996a). Thus, the parasitic

pressure from NPFWs is almost exclusively during the C phase.

The developmental process of dioecious fig tree species is slightly different. In these

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species, male figs produce pollen and wasps but no seeds (no E phase), and female figs produce seeds but no wasps (no D phase) (Harrison and Yamamura 2003). Thus, the male figs are the target of the NPFWs and female figs only rarely (Wu et al. 2013) which could lead to anatomical differences between male and female figs. In addition, female trees produce usually less figs than male trees (Bain et al. 2014) providing less oviposition sites for NPFWs. Also, dioecious fig species host a reduced diversity of NPFW species compared to monoecious fig species (Kerdelhué and Rasplus 1996b).

Draft 2.2 Plant material

Twenty-six species (29 taxa in total counting the varieties and subspecies) of native or introduced (one single species: F. altissima) fig trees have been recorded in Taiwan (Bain et al. 2015). The proportion of dioecious species is particularly high (20 of the 26 species) while worldwide about half of the Ficus species are dioecious. In Taiwan, fig tree species are widely distributed from the coastal areas (F. pedunculosa var. mearnsii) to the montane forests up to 1000 m above sea level (F. erecta var. beecheyana), with several species, such as F. benjamina var. bracteata and F. pedunculosa, restricted to the southern tropical part of the island (Tzeng 2004).

More than 200 figs from 21 fig tree species (one species with two varieties: sample sizes per species in Fig. 2 and 3) from six subgenera were collected on Taiwan Island and

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Orchid Island (southeast of Taiwan Island) from September 2013 to September 2015

(Table S1 in supplementary material). In Northern Taiwan, an exotic species, F. altissima,

which has a pollinating wasp in Taiwan, was sampled on the National Taiwan University

campus and in Da’an Forest Park in Taipei City. Figs of F. caulocarpa, F. subpisocarpa,

and F. microcarpa were sampled at various stages of development in order to study the

developmental changes in detail whereas figs at the C phase were sampled for all the

remaining studied fig species.

Draft 2.3. Sample preparation

In order to measure the fig diameter and fig wall thickness, the figs were cut

longitudinally through the ostiole and peduncle (Fig. 1). For ideal fixation, the figs were

dissected from the widest part into small blocks (less than 4 mm long and 5 mm wide).

For paraffin sections, the samples were first fixed either in FPGA solution

(formalin/propionic acid/glycerol/95% ethanol/distilled water at 1:1:3:7:8) or 0.5%

glutaraldehyde in 0.1 M phosphate buffer. In order to reduce the potential for the

formation of artefacts, samples collected in the field were directly fixed in 0.5%

glutaraldehyde in 0.1 M phosphate buffer. The tissue blocks were then washed in 50%

alcohol or 0.1 M phosphate buffer and dehydrated in a tert-butyl alcohol series. After

dehydration, the samples were infiltrated and embedded in paraffin (Paraplast; Leica

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Biosystems), and then sectioned using a rotary microtome (HM320, Microm) mounted with disposable microtome blades (Leica Biosystems). The thickness of the sections was

10–14 μm depending on the hardness of the tissues and was 20 μm for histochemical tests.

2.4 Staining and histochemical tests

The following staining methods were performed on deparaffinized sections: (a) Toluidine

Blue O in 1% borax as the metachromatic stain (Mercer 1963), (b) Fast Green–Safranin Draft O for differentiating the tissue structure (Sass 1958), (c) phloroglucinol in 6 N HCl for lignin localization (O’Brien and McCully 1981), and (d) ferric chloride solution with sodium carbonate to highlight tannins (Johansen 1940). The freehand sections fixed with

70% ethanol were treated with Sudan black B for staining the latex (Johansen 1940).

Positive control staining using locally known plant species samples was conducted for the aforementioned staining methods.

2.5 Imaging procedure and analyses

To measure the thickness of the fig wall (from the outer epidermis to the inner epidermis at the “equator” of the fig (Fig. 1)), the sections were photographed using a digital camera

(650D, Canon) mounted on a stereomicroscope (S8 APO, Leica Microsystems). For other

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freehand and paraffin sections, the slides were photographed using a Nikon D3 digital

camera mounted on a light microscope (DMRB, Leica Microsystems). The thickness of

the fig walls and each tissue type were measured using Fiji image processing software

(open source). Spearman rank correlation test was used to test the correlation between fig

wall thickness and fig diameter using SYSTAT 13 (Systat Software®, San Jose, CA,

USA).

3. Results

3.1. General fig wall structure Draft

Fig wall thickness was positively correlated with fig size (Fig. S1 in supplementary

material).

Among the 22 taxa (mean = 1640 ± 166 µm; n = 132), the figs of F. pumila var. pumila

had the thickest wall (mean = 8472 µm; n = 7), and those of F. ampelas had the thinnest

wall (mean = 529 µm; n = 4) (Fig. 2). Cross sections of the fig walls demonstrated that

they were composed of dermal (outer and inner epidermis), ground, and vascular tissues

(Fig. S2 in supplementary material). The outer epidermis may differentiate into trichomes

or lenticels. Laticifers and druses were common in the ground tissue of the fig wall and

were generally associated with vascular bundles.

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3.2. Fig wall classification

The fig walls (at C phase) were categorized into five types (Table 1) according to the ground tissue composition of the C phase fig wall (Fig. 4). The species and their fig wall types are summarized in Table 1.

Type I: The ground tissue was composed of only the regular parenchyma. This type was observed in the dioecious fig species (nine species from four subgenera). However, in two species (F. septica and F. benguetensis), only the female figs belong to the type I. On Draft the other hand, the male figs of these two species belong to the type III.

Type II: The ground tissue was characterized by aerenchyma (tissue with enlarged air spaces: Evans 2003) and a high density of laticifers. The aerenchyma was distributed between the parenchyma tissues near the outer and the inner epidermis. This type was observed only in two of the Taiwanese species (Table 1).

Type III: The ground tissue was characterized by sclerenchyma formation. Three subtypes were recognized according to the distribution of sclerenchyma cells: one-layered sclerenchyma (type IIIa), two-layered sclerenchyma (type IIIb), and scattered sclerenchyma cells (type IIIc). Type IIIc was observed only in F. nervosa figs.

Type IV: The parenchyma was characterized by abundant tannin deposition in the cells.

This type was observed only in three species belonging to two dioecious subgenera (Table

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1).

Type V: The parenchyma was characterized by the presence of both sclerenchyma and

tanniferous cells. This type was found mostly in monoecious species (75% of the species)

and once in the male figs of the dioecious species, F. pedunculosa var. mearnsii.

3.3. Developmental progress of fig walls in F. caulocarpa, F. subpisocarpa, and F.

microcarpa

The fig walls of F. caulocarpa and F. subpisocarpa were of type IIIb and their Draft developmental progress was similar (Table 1, Fig. 3). During A phase, the fig walls were

composed of only parenchyma. Tanniferous cells were present at the beginning of the A

phase, then quickly degraded after the fig peduncle elongated. At the end of the A phase,

a thin sclerenchyma layer developed near the outer epidermis, and a second thin

sclerenchyma layer formed when the figs became receptive (B phase). This two-layered

sclerenchyma structure was observed toward the end of maturation.

The fig wall of F. microcarpa was of type V and exhibited a distinct wall structure

until maturation. In comparison to F. caulocarpa and F. subpisocarpa, the young fig walls

(A phase) were composed of only parenchyma without tannins. At the end of A phase, a

thick tanniferous cell layer formed adjacent to the outer epidermis. In the B phase, a

sclerenchyma layer formed on the inner side of the tanniferous cell layer. After pollination

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(C phase), a second layer of sclerenchyma developed within the tanniferous cells, and the first sclerenchyma cell layer thickened. The two-layered sclerenchyma pattern was observed until the end of maturation. Nevertheless, the tannins disappeared in E phase.

3.4. Phylogeny of the fig wall types

In the 21 studied Taiwanese species, the different types of fig walls are distributed among the Ficus subgenera (Table 1, Fig. 5): types III and V in subgenus Urostigma; types I, III, and IV in subgenus Sycomorus; types I and III in subgenus Sycidium; types I, II, and III Draft in Synoecia; and types I and II in subgenus Ficus. In the six studied monoecious species, all the fig walls contained sclerenchyma tissues. In the subgenus Sycomorus subsection

Sycocarpus, the fig walls in male figs were of type IIIa and those in female figs were of type I.

4. Discussion

4.1. Thickened fig walls

The two species that efficiently suppressed NPFWs are F. erecta var. beecheyana and F. pumila (Tzeng et al. 2014; Bain et al. 2015), and the fig walls of both species are filled with aerenchyma. In general, aerenchyma is described as a response tissue that develops in the roots of flooded (Pimenta et al. 1998; Yamauchi et al. 2013). Moreover, it

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has been observed in fruits of some genera of the families Onagraceae (Eyde 1978) and

Asclepiadaceae (Kuriachen et al. 1992). The functions of aerenchyma include preventing

hypoxia and anoxia in flooded plants (Pimenta et al. 1998; Yamauchi et al. 2013),

increasing the buoyancy of fruit (Eyde 1978; Kuriachen et al. 1992). In Ficus, the floating

fig (F. cyathistipula in central Africa belonging to the subgenus Urostigma) has a spongy

fig wall (Berg and Wiebes 1992), but the effect of this spongy fig wall on NPFW species

is unknown. The efficient use of aerenchyma to thicken the fig wall at a lower cost and

weight has been observed at least twice in two distinct Ficus subgenera (F. erecta var. Draft beecheyana in subgenus Ficus and F. pumila in subgenus Synoecia). The fig wall

thickness should be more extensively investigated in relation to the associated

nonpollinating fig wasps and the duration of oviposition of these species.

4.2. Sophisticated fig walls

The studied species exhibited a great variety of fig wall anatomy from the simplest

parenchymatic wall (F. cumingii and F. tannoensis) to complex fig walls (F. benjamina

var. bracteata and F. microcarpa). Sclerenchymatous cell layers were slightly more

common than tanniferous cells. Hardened sclerenchymatous structures in infructescences

are often found in the Moraceae family (Corner 1962; Jacomassi et al. 2010; Oyama and

Souza 2011). Sclerenchyma can make up more than half of the fig wall (F. nervosa and

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F. pedunculosa var. pedunculosa) and up to two sclerenchyma layers have been found in four fig species. For other plant organs, such as leaves, sclerenchyma provides a certain amount of protection against herbivory (Peeters 2002; Rangasamy et al. 2009). But as all the studied fig species with sclerenchymatous cell layers have many nonpollinating fig wasp parasites (Bain et al. 2015), the hardened cell layers are probably not efficient against these wasps.

Seven fig species in this study have tanniferous cells in their fig walls. Among these seven species, three species have fig walls composed of only parenchyma with scattered Draft tanniferous cells (type IV). The presence of tannins may defend against fungal infection

(Holeski et al. 2009) and galling (Moctezuma et al. 2014) but no galling insects

(other than fig wasps) have been observed parasitizing the fig of any of the studied species in Taiwan (Bain A. personal observation).

On the other hand, F. caulocarpa, F. subpisocarpa, and F. microcarpa are parasitized by many nonpollinating fig wasp species (Bain et al. 2015). They belong to the same group (section Urostigma) within the Ficus classification but F. microcarpa is in subsection Conosycea whereas F. caulocarpa and F. subpisocarpa are in subsection

Urostigma (Berg and Corner 2005). The latter two species have very similar fig wall development whereas the development of the fig wall of F. microcarpa is slightly different. Other species from the subgenus Urostigma, F. ingens (section Urostigma,

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subsection Urostigma) and F. ottoniifolia (section Galoglychia, subsection Caulocarpae),

also have one sclerenchyma layer developing next to the outer epidermis. At maturity,

the figs of the three studied species, as well as F. ingens and F. ottoniifolia (Verkerke

1986; Baijnath and Naicker 1989), have two layers of sclerenchyma. Despite belonging

to the same subgenus, all the above species display some developmental differences but

compared to the other subgenera, they produce also the most complex fig wall. At this

point of the exploration of fig wall diversity, it is difficult to link any of the fig wall

anatomy to any ecological or behavioural characteristics. Draft Moreover, of the seven studied dioecious species, only two of them (F. septica and

F. benguetensis from the subgenus Sycomorus) differed in fig wall structure.

Sclerenchyma was observed only in the male figs (which are the only sex parasitized by

nonpollinating fig wasps). We can hypothesize that sclerenchyma in male figs may be a

consequence of the parasitism from nonpollinating fig wasps or from any other larger

parasites.

4.3. Phylogeny of the fig wall structure

The distribution of fig wall types across the Taiwanese Ficus species is not strongly

associated with their . Nevertheless, some patterns can be identified. First, some

features exist only in some groups: the type IIIb fig wall in subgenus Urostigma and the

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type IIIc fig wall in subgenus Pharmacosycea (only one species was sampled in this subgenus). Second, similar structures, such as aerenchyma or scattered tannin cells, appear within the subgenera Sycidium, Synoecia, and Ficus. In addition, the two species from the subgenus Sycomorus subsection Sycocarpus have a peculiar feature: the formation of sclerenchyma was observed only in the male figs.

Ficus erecta and F. pumila belong to two botanical lineages: subgenera Ficus and

Synoecia, respectively (Berg and Corner 2005). However, they have been grouped together in several molecular phylogenies in the subgenus Ficus (Jousselin et al. 2003; Draft Rønsted et al. 2005, 2008; Xu et al. 2011). Interestingly, this study presents them as the only two species with an aerenchymatous wall.

5. Conclusions

This study shows the anatomy of an overlooked structure of the fig: the fig wall. Simple fig walls (with only parenchyma) were almost as common as complex fig walls and many species exhibited layers of sclerenchyma, aerenchyma and tanniferous cells. The newly revealed structures of the fig wall display an unexpected diversity and the fig wall of other species from different regions should be investigated in order to gain a better understanding of the fig wall.

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Acknowledgements

We thank Seth Menser for the discussion about Ficus cyathistipula during the IXth

International fig symposium in Montpellier, France.

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Declaration of interest

The authors declare that they have no competing interests.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Table 1. Fig wall types of sampled Ficus species. The nonpollinating fig wasp (NPFW) species number updated from Bain et al. (2015).

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Subgenus Reproduction Species Wall type NPFW species Pharmacosycea Monoecious F. nervosa IIIc 2 F. altissima (exotic) V 223 F benjamina var. bracteata V 3 Urostigma Monoecious F. microcarpa V 23 F. caulocarpa IIIb 91 F. subpisocarpa IIIb 222 F. erecta var. beecheyana II 2 F. pedunculosa var. pedunculosa M: V Unknown Ficus Dioecious F. pedunculosa var. mearnsii F: I 1 F. tannoensis M: I 1 F. virgata IIIa 3 F. ampelas I 5 Sycidium Dioecious F. irisana F: I 6 F. cumingii F: I 1 F. tinctoriaDraft subsp. swinhoei M: I 7 F. benguetensis F: I, M: IIIa 4 Sycomorus Dioecious F. septica F: I, M: IIIa 6 F. variegata M: IV 4 F. pumila var. pumila II 0 F. punctata f. aurantiacea M: IV 1 Synoecia Dioecious F. sarmentosa var. nipponica F: I Unknown F. trichocarpa F: IV Unknown M: Male, F: Female 1 Chiang Y-P, unpublished data 2 Two additional species (Chen et al. 2013) 3 Data from www.figweb.org/Ficus/Subgenus_Urostigma/Section_Urostigma/Subsection_Conosycea/ Ficus_altissima.htm

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Figure captions

Fig. 1. Ficus pumila var. pumila. (A) Side view of the fig. (B) Top view of the fig from the ostiole. (C) Anatomy of the fig.

Fig. 2. Summary of C phase fig wall structure. From inner epidermis (0 mm thickness) to the outer epidermis. Total number of fig samples is 132 and the number indicates the number of samples used for each line. * indicates exotic species; ** scattered type.

Fig. 3. Summary of developmental progress of fig walls on F. caulocarpa, F. subpisocarpa, and F. microcarpa. From Inner epidermis (0 mm thickness) to the outer Draft epidermis. Numbers indicate the number of samples. The letters A to E represent the different development phases. Additional anatomical sections for the three species are in the supplementary material (Fig S2-S5).

Fig. 4. Anatomical sections of the five wall types. (A) Type I from a female F. ampelas;

(B) Type II from a female F. pumila var. pumila; (C) Type IIIa from a male F. virgata;

(D) Type IIIb from F. subpisocarpa; (E) Type IIIc from F. nervosa; (F) Type IV from a female F. trichocarpa and (G) Type V from F. microcarpa. A aerenchyma, IE inner epidermis, OE outer epidermis, P parenchyma, S sclerenchyma, T tanniferous cells, V vascular tissue. The scale bar is the same for all the photos.

Fig. 5. Taxonomical tree of the sampled species and their wall type (modified from Xu et al. 2011). * Ficus pumila belongs to the subgenus Synoecia (Berg and Corner 2005).

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Draft

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Figure 5

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