Viviane Gonçalves Leite

Finn Kjellberg

Rodrigo Augusto

Santinelo Pereira

Simone Pádua Teixeira

What makes a fig: insights from a comparative analysis of inflorescence morphogenesis in Moraceae

Keywords: anatomy, Brosimum, Castilla, Clarisia, development, Ficus, flower, Maclura, Morus, ontogeny

Background and Aims Moraceae, the family of mulberry and fig trees, displays small homogeneous flowers but extremely diverse inflorescences ranging from simple and branched to complex and condensed. Inflorescences also vary in flower organization in the receptacle, in the degree of flower condensation, and in the receptacle shape. Thus, the objective of the present study was to compare the inflorescence morphogenesis of Moraceae species, to investigate whether clades with a similar pollination mode share the same patterns of inflorescence development and at which developmental stages do we observe the key changes resulting in the diversified inflorescence architecture that culminates in the Ficus syconium.

• Methods Inflorescences at different developmental stages were sampled from Brosimum gaudichaudii, Castilla elastica, Clarisia ilicifolia, Ficus pertusa, Maclura tinctoria and Morus nigra and processed for surface and anatomical analyses.

• Key Results

The inflorescence morphogenesis of the studied species is highly variable. The shape of the inflorescence meristem (bulging, hemispheric or elongated), the initiation order and arrangement of flowers along the receptacle and the occurrence of bracts vary even between related species. This diversity originates early during inflorescence development. B. gaudichaudii, C. elastica and F. pertusa have flowers enclosed or immersed within the receptacle although inflorescences begin their development as flat and open structures, as occurs in the other three study species.

• Conclusion

The comparison of the inflorescence morphogenesis in Moraceae species allows us to infer that evolutionary ontogenetic changes driven by pollinators culminate in the enclosure of flowers inside the receptacle, as occurs in the Ficus syconium.

INTRODUCTION

The diversity of floral morphology and inflorescence architecture within angiosperms illustrates the extreme evolutionary plasticity of their reproductive structures [START_REF] Harder | The interplay between inflorescence development and function as the crucible of architectural diversity[END_REF]. Although floral morphological diversity has been described for more than two centuries (Baker, 1983) and characterized since [START_REF] Darwin | On the various contrivances by which British and foreign orchids are fertilised by insects[END_REF][START_REF] Darwin | The different forms of flowers on plants of the same species[END_REF], inflorescence morphology has received much less attention [START_REF] Wyatt | Inflorescence architecture: how flower number, arrangement, and phenology affect pollination and fruit-set[END_REF][START_REF] Harder | Beyond floricentrism: the pollination function of inflorescences[END_REF][START_REF] Prusinkiewicz | Evolution and development of inflorescence architectures[END_REF][START_REF] Harder | The interplay between inflorescence development and function as the crucible of architectural diversity[END_REF]. This is surprising since, taken together, flowers and inflorescences often constitute functional units for pollination, fruit development, attraction of frugivores, and seed dispersal [START_REF] Harder | The interplay between inflorescence development and function as the crucible of architectural diversity[END_REF]. Inflorescence shape is constrained by developmental, genetic and environmental factors [START_REF] Tucker | The inflorescence: Introduction[END_REF][START_REF] Singer | Inflorescence architecture: A developmental genetics approach[END_REF] that will condition its response to the selective pressures exerted by pollinators [START_REF] Ishii | Combined effects of inflorescence architecture, display size, plant density and empty flowers on bumble bee behaviour: experimental study with artificial inflorescences[END_REF]. Inflorescences are classified according to several criteria, such as ontogeny, shape and size of the axis (= receptacle), and occurrence and size of a peduncle (see [START_REF] Rickett | The classification of inflorescences[END_REF][START_REF] Weberling | Typology of inflorescences[END_REF][START_REF] Weberling | Inflorescence structure in primitive angiosperms[END_REF][START_REF] Weberling | Morphology of flowers and inflorescences[END_REF]Bell, 1991;Greyson, 1994;[START_REF] Weberling | Die Infloreszenzen. Typologie und Stellung im Aufbau des Vegetationskörpers[END_REF][START_REF] Tucker | The inflorescence: Introduction[END_REF][START_REF] Singer | Inflorescence architecture: A developmental genetics approach[END_REF][START_REF] Prenner | The key role of morphology in modelling inflorescence architecture[END_REF][START_REF] Endress | Disentangling confusions in inflorescence morphology: Patterns and diversity of reproductive shoot ramification in angiosperms[END_REF][START_REF] Claßen-Bockhoff | Towards an ontogenetic understanding of inflorescence diversity[END_REF][START_REF] Kirchoff | Inflorescences: concepts, function, development and evolution[END_REF].

Moraceae, the family of mulberry and fig trees, displays small homogeneous flowers but extremely diverse inflorescences ranging from simple and branched to complex and condensed [START_REF] Berg | Moreae, Artocarpeae, Dorstenia (Moraceae). With introductions to the family and Ficus and with additions and corrections to Flora Neotropica Monograph 7[END_REF][START_REF] Datwyler | On the origin of the fig: Phylogenetic relationships of Moraceae form ndhF sequences[END_REF][START_REF] Ribeiro | Studies on phylogeny, taxonomy and evolution of reproductive characters in Moraceae Gaudich[END_REF]. Inflorescences also vary in terms of the flower organization in the axis/receptacle, in the form and degree of condensation/union of the flowers, and in the shape of the receptacle, which is reflected in the diversity of inflorescence types and shapes (racemes, spikes, cyme, syconium, head, urceolate receptacles, [START_REF] Clement | Morphological Evolution in the Mulberry Family (Moraceae)[END_REF] and even in the types of infructescence [START_REF] Berg | Moreae, Artocarpeae, Dorstenia (Moraceae). With introductions to the family and Ficus and with additions and corrections to Flora Neotropica Monograph 7[END_REF]. Such extreme variation is accompanied by a wide variation in breeding systems within the family (monoecious, dioecious, andromonoecious, gynodioecious), and inflorescences may show pistillate and/or staminate flowers, even in monoecious species [START_REF] Berg | Moreae, Artocarpeae, Dorstenia (Moraceae). With introductions to the family and Ficus and with additions and corrections to Flora Neotropica Monograph 7[END_REF][START_REF] Berg | Moraceae genera other than Ficus[END_REF][START_REF] Ribeiro | Studies on phylogeny, taxonomy and evolution of reproductive characters in Moraceae Gaudich[END_REF][START_REF] Clement | Morphological Evolution in the Mulberry Family (Moraceae)[END_REF][START_REF] Zerega | Delimitation of the new tribe Parartocarpeae (Moraceae) is supported by a 333-gene phylogeny and resolves tribal level Moraceae taxonomy[END_REF]. Moraceae encompasses approximately 1,100 species, 39 genera [START_REF] Clement | Morphological Evolution in the Mulberry Family (Moraceae)[END_REF][START_REF] Berg | Moraceae genera other than Ficus[END_REF][START_REF] Berg | Flora Malesiana precursor for the treatment of Moraceae 8: Other genera than Ficus[END_REF] and seven tribes (Artocarpeae, Castilleae, Ficeae, Dorstenieae, Maclureae, Moreae and Parartocarpeae, [START_REF] Zerega | Delimitation of the new tribe Parartocarpeae (Moraceae) is supported by a 333-gene phylogeny and resolves tribal level Moraceae taxonomy[END_REF]. Thus, its representatives are excellent models for studies of the functional diversity of reproductive structures.

Investigating their inflorescence ontogeny may provide insights into selective pressures that shaped the floral moracean structures.

Inflorescence architecture in Moraceae may vary within and among taxonomic levels (i.e., species, genera and tribes) [START_REF] Berg | Moreae, Artocarpeae, Dorstenia (Moraceae). With introductions to the family and Ficus and with additions and corrections to Flora Neotropica Monograph 7[END_REF][START_REF] Berg | Moraceae genera other than Ficus[END_REF][START_REF] Ribeiro | Studies on phylogeny, taxonomy and evolution of reproductive characters in Moraceae Gaudich[END_REF][START_REF] Clement | Morphological Evolution in the Mulberry Family (Moraceae)[END_REF][START_REF] Zerega | Delimitation of the new tribe Parartocarpeae (Moraceae) is supported by a 333-gene phylogeny and resolves tribal level Moraceae taxonomy[END_REF]. In Maclureae, for example, the staminate inflorescences can be spicate, racemose or globose, whereas the pistillate inflorescence is globose [START_REF] Clement | Morphological Evolution in the Mulberry Family (Moraceae)[END_REF].

On the other hand, the syconium, an urn-shaped inflorescence that encloses diclinous (= unisexual) flowers densely arranged on the inner surface of the receptacle, with an apical opening called ostiole, is exclusive to Ficus [START_REF] Clement | Morphological Evolution in the Mulberry Family (Moraceae)[END_REF]. Similarly, discoid inflorescences, containing diclinous flowers immersed in the receptacle, are restricted to Dorstenieae [START_REF] Berg | Moreae, Artocarpeae, Dorstenia (Moraceae). With introductions to the family and Ficus and with additions and corrections to Flora Neotropica Monograph 7[END_REF][START_REF] Clement | Morphological Evolution in the Mulberry Family (Moraceae)[END_REF]. Despite the diversity of inflorescence architecture and richness in species [START_REF] Berg | Flora Malesiana precursor for the treatment of Moraceae 8: Other genera than Ficus[END_REF], few studies on the inflorescence development of this family are available (see Baillon, 1861;Golenkin, 1894;Bernbeck, 1932;[START_REF] Moncur | Floral ontogeny of the Jackfruit, Artocarpus heterophyllus Lam. (Moraceae)[END_REF].

From a series of morphological traits and published pollination observations, we may infer that Moreae is generally wind pollinated, Artocarpeae is pollinated by gall midges that breed in staminate inflorescences, Maclureae is wind pollinated, Dorstenieae can be pollinated by flies that breed in the inflorescences or wind pollinated, Castilleae is pollinated by thrips that breed in staminate inflorescences, and Ficeae is pollinated by agaonid wasps that breed in the urnshaped inflorescences [START_REF] Berg | The genus Dorstenia (Moraceae)[END_REF][START_REF] Sakai | Artocarpus (Moraceae)-gall midge pollination mutualism mediated by a male-flower parasitic fungus[END_REF][START_REF] Sakai | Thrips pollination of androdioecious Castilla elastica (Moraceae) in a seasonal Tropical forest[END_REF][START_REF] Kjellberg | Biology, ecology and evolution of fig pollinating wasps (Chalcidoidea: Agaonidae)[END_REF][START_REF] Taylor | High-speed pollen release in the white mulberry tree, Morus alba L[END_REF][START_REF] Araújo | From anthesis to diaspore dispersal: reproductive mechanisms of rare herbaceous Moraceae species endemic to Brazil[END_REF]. Hence, available data suggest that Moraceae are either wind pollinated or are involved in a series of biologically varied brood site pollination mutualisms. We propose that different types of brood site pollination mutualisms select for divergent inflorescence structures. Comparing the development of such diversified inflorescences may provide some insights into the morphogenic control of floral traits.

Thus, the objective of the present study was to compare the inflorescence morphogenesis of Moraceae species in order to investigate the role of pollination mode on the inflorescence development that culminates in the Ficus syconium. For that purpose, we compared the inflorescence development of wind-and insect-pollinated species, seeking to key out developmental changes associated with the inflorescence architecture.

MATERIALS AND METHODS

Six species representing six of the seven recognized tribes were sampled: Morus nigratribe Moreae (elongated raceme, oval spike), Clarisia ilicifolia -tribe Artocarpeae (spike, head), Maclura tinctoria -tribe Maclureae (elongated spike, globose head), Brosimum gaudichaudii -tribe Dorstenieae (globose head), Castilla elastica -tribe Castilleae (bivalvate, urceolate, discoid heads), and Ficus pertusa -tribe Ficeae (urn-shaped syconium) (Figure 1, Table 1). Representatives of C. ilicifolia, M. tinctoria and B. gaudichaudii are naturally found in Brazilian vegetational formations [START_REF] Berg | Moreae, Artocarpeae, Dorstenia (Moraceae). With introductions to the family and Ficus and with additions and corrections to Flora Neotropica Monograph 7[END_REF]. M. nigra, F. pertusa and C. elastica were introduced in Brazil, and naturally occur in northern warm-temperate regions (M. nigra - [START_REF] Berg | Moraceae genera other than Ficus[END_REF] and in Central America (F. pertusa and C. elastica [START_REF] Berg | Moreae, Artocarpeae, Dorstenia (Moraceae). With introductions to the family and Ficus and with additions and corrections to Flora Neotropica Monograph 7[END_REF]. About 50-150 inflorescences at different stages of development were collected and analyzed for each species. At least two individuals were sampled for each monoecious species and four for the dioecious species. For Castilla elastica, an androdioecious species, the pistillate inflorescences were collected on a tree producing both pistillate and staminate inflorescences (= cosexual tree) and the staminate inflorescences were collected from a tree producing only staminate inflorescence (= male tree). The plants were georeferenced, and the vouchers were deposited in the MBLM, SPFR and RB Herbaria (Table 1).

Samples were fixed in buffered formalin [START_REF] Lillie | Histopathologic technic and practical histochemistry[END_REF] or Karnovsky's solution (0.075 mol/M in phosphate buffer, pH 7.2-7.4) [START_REF] Karnovsky | A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy[END_REF] for 24 h, gradually dehydrated in an ethanol series and stored in 70% ethanol. They were dissected and prepared for surface analysis by scanning electron microscopy (SEM) and for anatomical analysis by light microscopy (LM). For SEM, samples were critical point dried in a Balzers CPD 030 dryer, mounted on aluminium stubs with colloidal carbon, coated with gold in a Bal Tec SCD 050 sputter coater and observed with Jeol JSM 6610LV and Zeiss EVO 50 scanning electron microscopes. For LM, samples were embedded in historesin [START_REF] Gerrits | The Application of Glycol Methacrylate in Histotechnology; Some Fundamental Principles[END_REF] and transversely and longitudinally cut into 2-5 μm thick sections with a rotary microtome (Leica RM 2245). Sections were stained with 0.05% Toluidine Blue [START_REF] O'brien | Polychromatic staining of plant cell walls by Toluidine Blue O[END_REF], mounted in synthetic resin [START_REF] Gerlach | Botanische Mikrotechnik, eine Einführung[END_REF] and observed under a Leica DM 5000B light microscope. Photomicrographs were taken with a Leica DM 5000 B microscope coupled to a Leica DFC 295 digital camera.

The terminology for bracts was based on [START_REF] Berg | Evolution, systematics, and fossil history of the Hamamelidae 2[END_REF], with modifications. The bracts are classified as follows according to the order in which they are initiated during the development of the inflorescence: bract of the inflorescence receptacle (first to arise); interfloral bract (arising among flowers); involucral bract (arising around the receptacle in Castilla and some species of Ficus); orobract (arising at the syconium orifice in Ficus). The interfloral bracts are further classified according to shape into simple or peltate [START_REF] Ribeiro | Studies on phylogeny, taxonomy and evolution of reproductive characters in Moraceae Gaudich[END_REF].

The terminology for inflorescence type was based on [START_REF] Troll | Organisation und Gestalt im Bereich der Blute[END_REF], [START_REF] Weberling | Morphology of flowers and inflorescences[END_REF] and [START_REF] Endress | Disentangling confusions in inflorescence morphology: Patterns and diversity of reproductive shoot ramification in angiosperms[END_REF]. The inflorescence is characterized as a raceme if all first and second order axes are elongated; as a spike, if the first order axis is elongated and the second order axis is short; and as a head, if all axes are short in the branching region. In this last case, the axis is reduced and can be conical and wide, discoid and plane, or flashy and in some cases it is surrounded by bracts.

The definitions for breeding systems included in the table 2 follow Ronse De Craene (2010), [START_REF] Sakai | Thrips pollination of androdioecious Castilla elastica (Moraceae) in a seasonal Tropical forest[END_REF] and [START_REF] Richards | Plant Breeding Systems[END_REF]: a dioecious species exhibits staminate and pistillate flowers on different plants; a monoecious species exhibits staminate and pistillate flowers in the same plant; in an androdioecious species coexist plants with staminate flowers and plants with both staminate and pistillate flowers.

RESULTS

The studied species are dioecious, monoecious or androdioecious. The inflorescence varies in terms of type (raceme, spike, head and syconium), format of the receptacle (elongated, oval, discoid, urceolate) and number of flowers per inflorescence (1 to ~ 190) (Table 2, Figure 1).

The inflorescence morphogenesis is also highly variable. No species pair followed the same inflorescence morphogenesis (Table 3, Figure 2). Variation was mainly observed in the shape of the inflorescence meristem (bulging, with a central depression, hemispheric, elongated), in the emergence order and arrangement of flowers along the inflorescence receptacle (Table 3, Figure 2), and in the occurrence of associated bracts.

Organography and inflorescence morphogenesis may vary within species between staminate and pistillate inflorescences, as in Maclura tinctoria and Morus nigra (Tables 2 and3).

Maclura tinctoria, Clarisia ilicifolia and Morus nigra

Organography (Table 2): The species of this subclade are dioecious, have raceme, spike or head types of inflorescences (Figure 1A-F), 30-150 sessile or pedicellate staminate flowers and 15-50 sessile or pedicellate pistillate flowers (Table 2). Receptacle bracts are occasionally found and interfloral bracts are generally found (Table 2).

Inflorescence development (Table 3): One to three inflorescence meristems can be formed in a bract axil, next or not to a vegetative meristem. The inflorescence meristem is in general elliptic (Figure 3). Its distal portion forms a horizontally elongated receptacle where the floral meristems first arise on the lateral sides (and/or adaxial side), from the base to the apex (Figure 4). In some cases, the distal portion of the inflorescence meristem forms bracts before the emergence of floral meristems (Figure 4). After the emergence of floral meristems and/or bracts, the proximal portion of the inflorescence meristem elongates and forms a peduncle that can be small or large (figures 4, 5). The developing flowers can form or not a pedicel (Figure 5).

Brosimum gaudichaudii, Castilla elastica and Ficus pertusa

Organography (Table 2): The species of this subclade are monoecious or androdioecious, have raceme or syconium types of inflorescences (Figure 1G-L), 9-150 pedicellate staminate flowers and 1-190 pedicellate/sessile pistillate flowers (Table 2). Receptacle bracts are always found and interfloral bracts do not occur in general (Table 2).

Inflorescence development (Table 3): The inflorescence meristem is rounded and subtended by a bract (Figure 6). Its distal portion forms a concave receptacle where bracts arise, covering the developing inflorescence (Figure 7). Next, the receptacle thickens, remaining flat (B. gaudichaudii) or forms a central depression with margins largely expanded (Figure 7). The margins of the receptacle do not close and the receptacle acquires a bivalvate or discoid format (C. elastica) or the margins completely enclose the inflorescence resulting in an urceolate form (F. pertusa) (Figures 7,8;suppl. 1). The floral meristems emerge in the center of the receptacle or asynchronously (Figure 7). Finally, the proximal portion of the inflorescence meristem forms a peduncle (Figure 8).

DISCUSSION

We present data for two monoecious species whose two types of floral morphs (staminate and pistillate) are found in the same inflorescences (B. gaudichaudii and F. pertusa), data for three dioecious species with floral morph types organized into two types of inflorescences (staminate and pistillate) very different from each other (C. ilicifolia, M. tinctoria, M. nigra), and for an androdioecious species, in which one type of plants present the two types of flowers in separate inflorescences (staminate and pistillate) and the other type of plants only presents inflorescences with staminate flowers. In the three dioecious species, staminate and pistillate inflorescences are very different from each other (see Table 2), possibly due to their functions in the release (staminate) and reception (pistillate) of pollen grains and for insect pollinated species in the attraction and/or breeding of the pollinators. In the case of C. elastica, the androdioecious species, there is variation in the receptacle shape and in the occurrence of bracts between the staminate inflorescences found on the two tree types (see Table 2). This means that the function of inflorescences containing staminate flowers on "cosexual" plants differs from that of inflorescences on "male" trees. We suggest the hypothesis that they may serve to attract insects to plants bearing pistillate inflorescences rather than serving for pollinator breeding and pollen dispersal.

What makes a fig? Transitions in the inflorescence structure of Moraceae species

Among the species studied here, Brosimum gaudichaudii and Castilla elastica belong to clades closely related to the tribe of Ficus pertusa (see Clement and Weiblen, 2009, Figure 12;[START_REF] Zerega | Delimitation of the new tribe Parartocarpeae (Moraceae) is supported by a 333-gene phylogeny and resolves tribal level Moraceae taxonomy[END_REF][START_REF] Clement | Evolution and classification of figs (Ficus, Moraceae) and their close relatives (Castilleae) united by involucral bracts[END_REF]. In these three species, inflorescences begin their development as flat and open structures (present study), suggesting that selective pressures, exerted by pollinators or by herbivores and seed predators have selected to enclose the flowers inside the inflorescence, culminating with the syconium of Ficus. The urn-shaped receptacle of Ficus is closed at the apex by orobracts forming a narrow passage (ostiole) so that the numerous internally distributed diclinous flowers can only be pollinated by fig wasps and pollen can only be collected from a syconium by the wasps born inside it (Hymenoptera, Agaonidae), characterizing an obligatory mutualism between fig trees and fig wasps [START_REF] Moe | Rapid evolution of pollinator-mediated plant reproductive isolation[END_REF]. The orobracts serve at least two functions in Ficus: enclosing the syconium and emitting receptive syconium odors that will attract pollinators [START_REF] Souza | Diversity of fig glands is associated with nursery mutualism in fig trees[END_REF][START_REF] Hu | Similitudes and differences between two related species of Ficus species in the synthesis by the ostiole of odors attracting their host-specific pollinators: a transcriptomic based investigation[END_REF]. In some species, epidermal cells of ostiolar bracts produce an exudate that plugs the entry of the syconium after pollinator entry [START_REF] Machado | Secretory structures at syconia and flowers of Ficus enormis (Moraceae): a specialization at ostiolar bracts and the first report of inflorescence colleters[END_REF].

We may propose here that they perform one more function, molding the development of the flowers by filling up the future syconium cavity, thus controlling flower development, as suggested for Ficus sur [START_REF] Verkerke | Flower development in Ficus sur Forsskal (Moraceae)[END_REF]).

An evident transitional stage of this enclosure is observed in the three different types of inflorescence of Castilla elastica: (1) discoid head, condensed inflorescence with a receptacle almost totally enclosed by marginal involucral bracts and united flowers (pistillate inflorescence); (2) urceolate "fig-like" head inflorescence with an apical orifice covered with imbricate bracts (complementary staminate inflorescences found in "cosex" trees), (3) bivalvate inflorescence enclosed by marginal bracts (staminate inflorescences found in male trees). These forms of inflorescences do not completely enclose the flowers, but, even so, staminate inflorescences only expose the staminate flowers late during inflorescence development. Castilla is pollinated by thrips [START_REF] Sakai | Thrips pollination of androdioecious Castilla elastica (Moraceae) in a seasonal Tropical forest[END_REF] and, as proposed for Ficus, we hypothesize that pistillate flowers are not the main structures involved in pollinator attraction, but staminate inflorescences on "cosexual" plants could also play that role.

In Brosimum gaudichaudii [START_REF] Jacomassi | Morfoanatomia e histoquímica de órgãos reprodutivos de Brosimum gaudichaudii (Moraceae)[END_REF] present study) and some Dorstenia species [START_REF] Araújo | From anthesis to diaspore dispersal: reproductive mechanisms of rare herbaceous Moraceae species endemic to Brazil[END_REF], the inflorescence receptacle is expanded and thick but does not enclose pistillate or staminate flowers. However, these flowers are almost totally immersed in the receptacle and thus are hidden in the inflorescence. Little is known about pollination in the tribe Dorstenieae that includes Brosimum and Dorstenia [START_REF] Berg | Moraceae genera other than Ficus[END_REF]. Brosimum alicastrum is anemophilous, but it could be atypical in this respect within the genus [START_REF] Berg | Moreae, Artocarpeae, Dorstenia (Moraceae). With introductions to the family and Ficus and with additions and corrections to Flora Neotropica Monograph 7[END_REF]. Dorstenia has been better studied. Beetles [START_REF] Hoen | Pollen morphology of the tribe Dorstenieae (Moraceae)[END_REF][START_REF] Berg | The genus Dorstenia (Moraceae)[END_REF] and flies [START_REF] Berg | The genus Dorstenia (Moraceae)[END_REF][START_REF] Araújo | From anthesis to diaspore dispersal: reproductive mechanisms of rare herbaceous Moraceae species endemic to Brazil[END_REF] were found visiting the flowers immersed in the cup-or flask-shaped receptacle of the cenanthum (Golenkin, 1894). An important aspect here is the strong protogyny within the inflorescence. The female phase may last 14-15 days and the male phase may last over 30 days with limited, if any, overlap [START_REF] Araújo | From anthesis to diaspore dispersal: reproductive mechanisms of rare herbaceous Moraceae species endemic to Brazil[END_REF]. Hence, the offspring of an insect ovipositing during the female phase may emerge when the inflorescence is still producing pollen. This phenology was probably a prerequisite for the occurrence of the mutualistic pollination system between Ficus and Agaonid wasps.

The enclosure and protection of flowers by receptacles whose margins elongate and completely or partially enclose the inflorescence have several consequences for the pollination of this entomophilous group of plants. Pollinating insects (flies in D. ariifolia, thrips in C. elastica and fig wasps in species of F. pertusa and other species) are attracted to the inflorescence by odor glands [START_REF] Souza | Diversity of fig glands is associated with nursery mutualism in fig trees[END_REF] located externally on the receptacle and in the various types of bracts found at the inflorescence closure sites, such as the ostiole in Ficus [START_REF] Machado | Secretory structures at syconia and flowers of Ficus enormis (Moraceae): a specialization at ostiolar bracts and the first report of inflorescence colleters[END_REF][START_REF] Souza | Diversity of fig glands is associated with nursery mutualism in fig trees[END_REF].

Glands secreting nectar (extrafloral nectary), mucilage (colleters), phenolic compounds (idioblasts or secretory epidermis) and latex (laticifers) can also be found [START_REF] Machado | Secretory structures at syconia and flowers of Ficus enormis (Moraceae): a specialization at ostiolar bracts and the first report of inflorescence colleters[END_REF][START_REF] Souza | Diversity of fig glands is associated with nursery mutualism in fig trees[END_REF] Marinho and Teixeira 2019) on both surfaces of the receptacle, with functions concerning the protection of the inflorescence against radiation and herbivores, lubrication of the developing floral organs and short-distance attraction of pollinators. These insects use inflorescences for oviposition and offspring protection ("nursery pollination") and even for mating, while transferring pollen passively (C. elastica, [START_REF] Zerega | Pollination in the New Guinea endemic Antiaropsis decipiens (Moraceae) is mediated by a new species of thrips, Thrips antiaropsidis sp. nov. (Thysanoptera: Thripidae)[END_REF] or actively (F. pertusa, [START_REF] Frank | The behavior and morphology of the fig wasps Pegoscapus assuetus and P. jimenezi: Descriptions and suggested behavioral characters for phylogenetic studies[END_REF][START_REF] Kjellberg | Pollination mode in fig wasps: the predictive power of correlated traits[END_REF] between flowers.

In addition, these inflorescences acquire the appearance of a floral unit [START_REF] Berg | Olmedieae Brosimeae (Moraceae)[END_REF], i.e., a pseudanthous inflorescence. Pseudanthy is correlated with the discoid and urceolate shapes of receptacles especially if they are involucrate or bear marginal bracts (e.g. Castilleae and Dorstenieae, [START_REF] Berg | Urticales, their Differentiation and Systematic Position[END_REF]. Reduction of such inflorescences can also result in uniflorous pistillate inflorescences, as is the case for Ficus oleifolia and Pereba humilis [START_REF] Berg | Urticales, their Differentiation and Systematic Position[END_REF][START_REF] Berg | Moraceae genera other than Ficus[END_REF].

The racemes, spikes and heads of Maclura tinctoria, Clarisia ilicifolia and Morus nigra

Unlike Brosimum gaudichaudii, Castilla elastica and Ficus pertusa, the inflorescences of Clarisia ilicifolia, Maclura tinctoria and Morus nigra are open, characterized as elongated spikes, globose heads or oval racemes (see Table 2). The raceme characterizes the pistillate inflorescence of M. nigra while the short floral pedicels characterize the pistillate inflorescence of M. nigra and staminate inflorescences of Clarisia ilicifolia and Maclura tinctoria as spikes. An inflorescence is characterized as a raceme if all first and second order axes are elongated, as a spike if the first order axis is elongated and the second axis is short, and as a head if all axes are short in the branching region. In the last case the axis is reduced and can be conical and wide, or even discoid (see Table 2) and plane, and can be surrounded by bracts [START_REF] Troll | Organisation und Gestalt im Bereich der Blute[END_REF][START_REF] Weberling | Morphology of flowers and inflorescences[END_REF][START_REF] Endress | Disentangling confusions in inflorescence morphology: Patterns and diversity of reproductive shoot ramification in angiosperms[END_REF]. Although the inflorescence meristem is shaped similarly in Clarisia ilicifolia, Maclura tinctoria and Morus nigra, the order of flower initiation along the receptacle is highly variable (acropetal, asynchronous, centrifugal, see Table 2) even in the same species, when we consider pistillate and staminate flowers (e.g., C. ilicifolia).

Morus nigra and Maclura tinctoria are two wind-pollinated species having inflexed filaments and anthers fitted in the sepals which is associated with an explosive mechanism of pollen release [START_REF] Pedersoli | Anatomy solves the puzzle of explosive pollen release in wind-pollinated urticalean rosids[END_REF]. This mechanism allows the release of pollen grains into the air at initial velocities exceeding Mach 0.7 [START_REF] Taylor | High-speed pollen release in the white mulberry tree, Morus alba L[END_REF]. We show that the ontogeny of the inflorescences differs between the two species for both staminate and pistillate inflorescences. The difference in staminate inflorescence ontogeny suggests functional convergence rather that identity by descent, in agreement with molecular results. Similarly, Castilla elastica is pollinated by thrips [START_REF] Sakai | Thrips pollination of androdioecious Castilla elastica (Moraceae) in a seasonal Tropical forest[END_REF] as is Antiaropsis decipiens [START_REF] Zerega | Pollination in the New Guinea endemic Antiaropsis decipiens (Moraceae) is mediated by a new species of thrips, Thrips antiaropsidis sp. nov. (Thysanoptera: Thripidae)[END_REF] within the Castilleae. Clarisia ilicifolia is probably pollinated by insects, as demonstrated for Artocarpus integer [START_REF] Sakai | Artocarpus (Moraceae)-gall midge pollination mutualism mediated by a male-flower parasitic fungus[END_REF] and A. heterophyllus [START_REF] Gardner | A flower in fruit's clothing: pollination of jackfruit (Artocarpus heterophyllus, Moraceae) by a new species of gall midge, Clinodiplosis ultracrepidata sp. nov. (Diptera: Cecidomyiidae)[END_REF] within the tribe Artocarpeae. The development of staminate and pistillate inflorescences is strikingly different between Castilla and Clarisia. However, the analogy in pollination mode may be less marked in this comparison than in the case of Morus and Maclura. It is because in Artocarpeae the pollinating gall midges feed on fungi developing on staminate inflorescences, explaining the broad staminate inflorescence, while in Castilleae the pollinating thrips feed on pollen, explaining the higher density of anthers in its staminate inforescence [START_REF] Sakai | Artocarpus (Moraceae)-gall midge pollination mutualism mediated by a male-flower parasitic fungus[END_REF][START_REF] Sakai | Thrips pollination of androdioecious Castilla elastica (Moraceae) in a seasonal Tropical forest[END_REF][START_REF] Zerega | Pollination in the New Guinea endemic Antiaropsis decipiens (Moraceae) is mediated by a new species of thrips, Thrips antiaropsidis sp. nov. (Thysanoptera: Thripidae)[END_REF][START_REF] Gardner | A flower in fruit's clothing: pollination of jackfruit (Artocarpus heterophyllus, Moraceae) by a new species of gall midge, Clinodiplosis ultracrepidata sp. nov. (Diptera: Cecidomyiidae)[END_REF].

Another particularly important variable characteristic is related to the arrangement of the inflorescences along the shoot, which may be related to the presence of a vegetative meristem along with the reproductive meristem. This vegetative meristem can systematically produce a leafy shoot with short internodes, as in Maclura tinctoria and Morus nigra, or can sometimes continue its development into a leafy shoot, as in Brosimum gaudichaudii and Ficus pertusa. In some species of Ficus, such as F. carica [START_REF] Valdeyron | Sur le système génétique du figuier Ficus carica L. Essai d'interprétation évolutive[END_REF], or most species of the Pharmacosycea section (pers. obs.), a single inflorescence meristem (but sometimes two) is generally formed in association with a vegetative meristem. The presence of two syconia is the more general condition in Ficus. In a number of species belonging to diversified subgenera and sections, the vegetative meristem produces a reproductive branch that will bear many figs, with two or more at each internode. The transition from axillated figs to cauliflory has occurred many times, suggesting a simple ontogenic transition. In Ficus pertusa, however, we observe two syconia (see Fig. 1K) from the two inflorescence meristems, and the vegetative meristem centrally positioned in a triad of apices generally aborts (see Fig. 11B-F, H).

Ontogenic studies on inflorescence in cauliflorous Ficus species would permit the confirmation of the hypothesis of a direct association between this vegetative meristem and the production of cauliflorous branches.

The importance of bracts and peduncle for the inflorescence architecture of Moraceae

Our data show that bracts, whether of first or second order, peltate or simple, external or interfloral, originate in the inflorescence meristem and, therefore, are homologous in origin. The functions attributed to the bracts are also numerous. Bracts are cited as a protective organ either of the floral meristem or of the inflorescence meristem [START_REF] Berg | Olmedieae Brosimeae (Moraceae)[END_REF][START_REF] Berg | Moreae, Artocarpeae, Dorstenia (Moraceae). With introductions to the family and Ficus and with additions and corrections to Flora Neotropica Monograph 7[END_REF][START_REF] Clement | Morphological Evolution in the Mulberry Family (Moraceae)[END_REF] present study). In Ficus, the orobracts that line the ostiole play important roles regarding the closure of the syconium [START_REF] Berg | Olmedieae Brosimeae (Moraceae)[END_REF][START_REF] Berg | Moreae, Artocarpeae, Dorstenia (Moraceae). With introductions to the family and Ficus and with additions and corrections to Flora Neotropica Monograph 7[END_REF][START_REF] Mello-Filho | Morfologia de certos sicônios de Ficus (Moraceae)[END_REF], the attraction of pollinating fig wasps by compounds secreted by glands [START_REF] Machado | Secretory structures at syconia and flowers of Ficus enormis (Moraceae): a specialization at ostiolar bracts and the first report of inflorescence colleters[END_REF][START_REF] Souza | Diversity of fig glands is associated with nursery mutualism in fig trees[END_REF], and even the order of flower initiation in the syconium's receptacle because of the pressure they exert on the meristematic tissues [START_REF] Verkerke | Flower development in Ficus sur Forsskal (Moraceae)[END_REF][START_REF] Basso-Alves | Different ontogenetic processes promote dicliny in Ficus L. (Moraceae)[END_REF]present study). In Maclura, interfloral bracts are covered with globular or elliptical glands with a yellow content, suggesting a defense function against phytophagous insects [START_REF] Berg | Moreae, Artocarpeae, Dorstenia (Moraceae). With introductions to the family and Ficus and with additions and corrections to Flora Neotropica Monograph 7[END_REF]. In Ficus pertusa and Brosimum gaudichaudii, species that share the presence of a triad of meristems (a vegetative meristem among two reproductive ones), first-order external marginal bracts are observed only during the early development of inflorescences, when there is no contact between meristems in the triad, also indicating a protective function. The interfloral bracts of Moraceae may act as a barrier against phytophagous invasion [START_REF] Berg | Reproduction and evolution in Ficus (Moraceae): traits connected with the adequate rearing of pollinators[END_REF], as illustrated in Brosimum gaudichaudii and in Clarisia ilicifolia (pistillate inflorescence), in which the apices of the peltate bracts are imbricated. In contrast, the simple interfloral bracts have thin apices partially covering the floral meristems in Clarisia ilicifolia (staminate inflorescence) and Maclura tinctoria (staminate and pistillate inflorescences).

The presence and development of the inflorescence peduncle are already noticeable during the intermediate stages of inflorescence development in most of the studied species of Moraceae [START_REF] Moncur | Floral ontogeny of the Jackfruit, Artocarpus heterophyllus Lam. (Moraceae)[END_REF]present study). The presence of a peduncle ensures that the inflorescences remain evident in relation to the branches with leaves for a long period of time, thus promoting attractiveness for pollinators [START_REF] Marshall | Peduncle characteristics, inflorescence survival and reproductive growth of white clover (Trifolium repens L.)[END_REF]. Maclura tinctoria and Morus nigra show elongated, flexible and prominent staminate inflorescences exposed to airflow and susceptible to agitation, which facilitates the removal of pollen, since these species is wind-pollinated. The discoid and wide inflorescence of Castilla elastica is totally exposed in the branches when the flowers are receptive, since the leaves are deciduous.

CONCLUSION

Two general patterns of inflorescence development were observed in the study species. In Brosimum gaudichaudii, Castilla elastica and Ficus pertusa (subclade that encompasses the tribes Dorstenieae, Castilleae and Ficeae), the inflorescence receptacle is initially flat-discoid and, end up in an urceolate inflorescence that encloses the flowers. The inflorescence of Maclura tinctoria, Morus nigra and Clarisia ilicifolia (subclade formed by the tribes Moreae, Maclureae and Artocapeae), on the other hand, begins its development as an elongated structure that results on a spike, a globose head or an oval raceme.

The inflorescence morphogenesis is highly variable in Moraceae. No species pair followed the same inflorescence morphogenesis, even in cases of similar pollination mode. Such a great diversity of the inflorescence architecture remains a field of research to be explored. The main gap is still the comprehensive understanding of the structure and function of these inflorescences in the family, which could be filled by studies that relate morphological and ecological aspects to inflorescence development. Table 1. Information about the sampled species of Moraceae. Species are ranked according to their position in the phylogeny of [START_REF] Zerega | Delimitation of the new tribe Parartocarpeae (Moraceae) is supported by a 333-gene phylogeny and resolves tribal level Moraceae taxonomy[END_REF]

Fig. 1 .

 1 Fig. 1. Inflorescence morphology of Moraceae species. (A-B) Staminate (A) and pistillate (B)

Fig. 3 .

 3 Fig. 3. Early developmental stages of inflorescence of Maclura tinctoria (A-B), Clarisia ilicifolia (C-

Fig. 4 .

 4 Fig. 4. Intermediate developmental stages of inflorescence of Maclura tinctoria (A, B), Clarisia

Fig. 5 .

 5 Fig. 5. Final developmental stages (drawings) of inflorescence of Maclura tinctoria (A, B), Clarisia

Fig. 6 .

 6 Fig. 6. Early developmental stages of inflorescence of Brosimum gaudichaudii (A), Castilla elastica

Fig. 7 .

 7 Fig. 7. Intermediate developmental stages of inflorescence of Brosimum gaudichaudii (A), Castilla

Fig. 8 .

 8 Fig. 8. Final developmental stages of inflorescence (drawings) of Brosimum gaudichaudii (A),

  . IAC = Herbário Fanerogâmico e Criptogâmico do Instituto Agronômico de Campinas; MNHN = Muséum National d'Histoire Naturelle; RB = Herbário do Jardim Botânico do Rio de Janeiro; SPFR = Herbário do Departamento de Biologia da Faculdade de Filosofia Ciências e Letras de Ribeirão Preto da Universidade de São Paulo.

	Species	Tribe	Place of collection	Number of collector	Date of collection
	Maclura tinctoria (L.) D.Don ex Steud	Maclureae	Campus USP, Ribeirão Preto, Brazil	VG Leite 06 and ES Campos (SPFR); VG Leite 07 and ESCampos (SPFR)	IX/2012; II/2013; V/2013
	Clarisia ilicifolia (Spreng.) Lanj. & Rossberg	Artocarpeae	Serra do Cavalo, Picuã, Aracruz, Brazil	AP Fontana 7845 (MBLM)	X/2013
				VG Leite 03 and ES
	Morus nigra L.	Moreae	Campus USP, Ribeirão Preto, Brazil	Campos (SPFR); VG Leite 05 and ES	VII/2012; II/2013
				Campos (SPFR)
	Brosimum gaudichaudii Trécul	Dorstenieae	Sitio Tapera, Sacramento, MG, Brazil	VG Leite 01 and VG Leite (SPFR)	VIII/2012; X/2012; VI/2013
				VG Leite 04 (SPFR),
	Castilla elastica Sessé	Castilleae	Instituto Agronômico de Campinas, Brazil; Botanical Garden of Rio de Janeiro, Brazil	Carvalho 122 and J.F. Benedito (IAC); JC Gomes s/nº FF Moreira s/nº (RB (RB291386);	X/2012; III/2013; XII 2014
				422383)
	Ficus pertusa L. f.	Ficeae	Campus USP, Ribeirão Preto, Brazil	RAS Pereira et al. 127 (SPFR 9959)	X/2012; II/2013

Table 2 .

 2 Diagnostic characters of inflorescence morphology in the species studied. Symbols: + = presence, -= absence, n/a = non-available information.Light lines separated sister tribes, bold line separates basal groups of clades.

	Species	Breeding system/pollinator agent	Floral composition of the inflorescence	Type of inflorescence	Number of flowers	Receptacle shape	External receptacle bract	Simple interfloral bract	Peltate interfloral bract
	Morus nigra	dioecious/	staminate	raceme	30 (pedicellate)	elongated	-	-	-
		wind	pistillate	spike	53 (sessile)	oval	-	-	-
	Clarisia	dioecious/	staminate	spike	150 (sessile) elongated		+	+
	ilicifolia	gall midge	pistillate	head	15 (sessile)	globose	+		+
	Maclura	dioecious/	staminate	spike	150 (sessile) elongated	-	+	-
	tinctoria	wind	pistillate	head	40 (sessile)	globose	-	+	-
	Brosimum gaudichaudii	monoecious/ wind	staminate/pistillate	head	10 pistillate staminate/1	globose	+	-	+
			staminate ("male tree")	head	50 (sessile)	bivalvate	-(imbricate)	-	-
	Castilla elastica	androdioecious/ thrips	staminate ("cosexual tree")	head	400	urceolate discoid	+ (imbricate, interfloral)	-	n/a
			pistillate ("cosexual" tree)	head	16 (sessile)	with central	+ (imbricate)	-	-
						depression
					20
	Ficus pertusa	"monoecious fig wasps"	staminate/pistillate	syconium	staminate/ 190	urn (closed)	+ (orobracts)	n/a	n/a
					pistillate

Table 3 .

 3 Diagnostic characters of inflorescence development in the species studied. n/a = non-available information. Light lines separate sister tribes, bold lines separate basal groups of clades.

	Species	Floral composition of the inflorescence	Inflorescence meristem format	Number of inflorescence meristems	Occurrence of a vegetative meristem	Initiation order of flowers along the receptacle	Initiation order of structures in the inflorescence meristems
	Morus nigra	staminate pistillate	elongated elongated	1 2-3	yes sometimes	centripetal centripetal	receptacle, floral meristems, peduncle
	Clarisia ilicifolia	staminate pistillate	elongated elongated and elliptical	1 1	yes yes	acropetal asynchronous	receptacle, receptacle bracts, floral meristems, peduncle
	Maclura tinctoria	staminate pistillate	elliptical elliptical	1 1	yes yes	acropetal centrifugal	receptacle, simple interfloral bracts + floral meristems, peduncle
							receptacle, receptacle bracts,
	Brosimum gaudichaudii	staminate/pistillate	elliptical	2	yes	centrifugal (staminate flowers)	pistillate floral meristem, peltate interfloral bracts, staminate floral meristems,
							peduncle
	Castilla elastica	staminate ("male tree") staminate ("cosexual tree")	elliptical n/a	2 n/a	n/a n/a	asynchronous n/a	receptacle, receptacle bracts, staminate floral meristems + interstaminal bract n/a
		pistillate ("cosexual" tree)	elliptical	1	n/a	centrifugal	receptacle, receptacle bracts, pistillate floral meristems
							receptacle, receptacle bracts,
							orobracts, pistillate and
	Ficus pertusa	staminate/pistillate	elongated	2	yes	asynchronous	staminate floral meristems +
							peltate interfloral bracts,
							peduncle

ACKNOWLEDGEMENTS

We are grateful to Rodrigo Ferreira Silva (FFCLRP-USP), Maria Dolores Seabra Ferreira, José Augusto Maulin (FMRP-USP), Edimárcio da Silva Campos (FCFRP/USP), Juliana Villela Paulino (UFRJ), and André Paviotti Fontana for technical assistance, to Bruno Garcia Simões Favaretto for the drawings (figs. 1H', 2I, 3I, 4I, 5I, 6K, 7M, 8L, 9L, 10L, 11Q, 12 III), to Elettra Greene and Dewey for English revision.

FUNDING

This research was supported by Fapesp (grant numbers 2012/1544-8 and 2014/07453-3), CNPq (grant numbers 156638/2012-0 and 302806/2019-9) and CAPES (code number 001).