Morphological Variation and Floral Development of Major Clades in Urticaceae -A Focus on the Female Flowers

Morphological Variation and Floral Development of Major Clades in Urticaceae -A Focus on The Female Flowers

Jia Dong / August, 2016

Thesis submitted in partial fulfillment for the MSc in the Biodiversity and Taxonomy of Plants 1

Abstract

Urticaceae, as one of the largest wind-pollinated family, has a long taxonomic history. Molecular phylogeny of Urticaceae has suggested it as a monophyletic lineage in the order Rosales with four major clades (Urticeae, Elatostemeae, Boehmerieae and Cecropieae). However, the morphological character changes do not always corresponding with the molecular data, especially in the female variation. Morphological observation of species among 11 representative genera was examined and the floral development of five species covering three different tribes was investigated using SEM to understand the inter- and intra- familial relationships and the evolution trend of some important characters in Urticaceae. Dramatic variations occur in the female flowers of the family in terms of perianth merism, stigma and ovule morphology, floral trichomes and sexuality. All the species follow a similar floral developmental process where the gynoecium develops unequally and encloses the basal orthotropous ovule from one side leaving the opening at the other side and resulting in a grooved scar on the ovary wall. The carpel formation is analogical to that of other urticalean families but indicates a much more reduced and advanced stage.

Acknowledgements I would like to thank my supervisor, Dr. Louis Ronse De Craene, for proposing this project and giving me the opportunity to explore and expand the topic. He has offered great support and precious advices during my lab work as well as the writing. And I’m really grateful for his patience and kindness whenever I raised questions or came across difficulties. I also thank my co-supervisor, Dr. Alexandre Monro from the Royal Botanic Gradens, Kew, who has kindly provided me with most of the material and abundant information on the Urticaceae taxonomy and advised on my project enthusiastically throughout the whole period, especially during my stay in the Kew. Also thank the Kew for providing me convenient access to the herbarium and the samples from living collections. I would like to acknowledge Jenny Farrar, who kindly assisted me at the beginning of my dissection and warmly encouraged me. I have to thank Frieda Christie, as she offered training, advice and support upon all the lab work, especially the SEM. I’m grateful to all my lovely colleagues in the MSc course, especially Ludovica Santilli and Maria Papagianni, for your warm and supportive company and friendship. The project is fully supported by the Royal Botanical Garden, Edinburgh and all the staff I came across has been generous and willing to help us in many ways. In the end, I would never be able to express my gratitude adequately to my parents, who have been loving me and supporting me both mentally and financially ever since my birth.

Table of Contents

ABSTRACT ...... 1

ACKNOWLEDGEMENTS ...... 2

LIST OF FIGURES IN TEXT ...... 4

1 INTRODUCTION ...... 5

1.1 GENERAL MORPHOLOGY...... 5 1.2 CLASSIFICATION HISTORY ...... 6 1.3 PHYLOGENY AND EVOLUTIONARY IMPORTANCE OF URTICACEAE ...... 8 1.4 ECONOMIC IMPORTANCE ...... 10 1.5 AIM OF STUDY ...... 11

2 MATERIALS AND METHODS ...... 12

2.1 SAMPLE COLLECTION ...... 12 2.2 CRITICAL POINT DRYING ...... 12 2.3 SCANNING ELECTRON MICROSCOPE ...... 13

3 RESULTS ...... 14

3.1 VAIRABLE MORPHOLOGY OF THE FEMALE FLOWERS ...... 14 3.1.1 Urticeae ...... 14 3.1.2 Elatostemeae ...... 17 3.1.3 Boehmerieae ...... 19 3.1.4 Cecropieae ...... 24 3.2 FLORAL DEVELOPMENT OBSERVATION OF DIFFERENT TRIBES ...... 25 3.2.1 Urticeae ...... 25 3.2.2 Elatostemeae ...... 29 3.2.3 Boehmerieae ...... 36

4 DISCUSSION ...... 42

4.1 NATURE OF PERIANTH AND MERISTIC VARIATION ...... 42 4.2 SIGNIFICANT GYNOECIUM CHARACTERS ...... 44 4.3 TRICHOMES OF URTICACEAE FLOWERS ...... 46 4.4 EVOLUTION OF SEXUALITY IN URTICACEAE ...... 47 4.5 POLLINATION SYNDROMES OF URTICACEAE ...... 49 4.6 INDICATIONS OF INFRA-FAMILIAL AND INTERFAMILY RELATIONSHIPS OF URTICACEAE ...... 50

5 CONCLUSION ...... 52

REFERENCES ...... 54

APPENDIX ...... 58 4

List of Figures in Text Fig. 1 Phylogeny of Rosales ...... 8 Fig. 2 Phylogeny of four major tribes in Urticaceae ...... 9 Fig. 3 Morphological diversity of Urticaceae ...... 15 Fig. 4 Female variation in Urticeae ...... 16 Fig. 5 Female variation in Elatostemeae ...... 18 Fig. 6 Female variation in Boehmerieae (1) ...... 21 Fig. 7 Female variation in Boehmerieae (2) ...... 22 Fig. 8 Female variation in Boehmerieae (3) ...... 23 Fig. 9 Female variation in Cecropieae (Cecropia sp.) ...... 24 Fig. 10 Perianth initiation stages in Urtica dioica ...... 26 Fig. 11 Gynoecium developmental stages in Urtica dioica (1) ...... 27 Fig. 12 Gynoecium developmental stages in Urtica dioica (2) ...... 28 Fig. 13 Perianth developmental stages in Myriocarpa stipitata ...... 30 Fig. 14 Gynoecium developmental stages in Myriocarpa stipitata (1) ...... 31 Fig. 15 Gyneocium developmental stages in Myriocarpa stipitata ...... 32 Fig. 16 Perianth developmental stages in Pilea grandis...... 33 Fig. 17 Gynoecium developmental stages in Pilea grandis (1) ...... 34 Fig. 18 Gynoecium developmental stages in Pilea grandis (2) ...... 35 Fig. 19 Early developmental stages of bisexual flower in Parietaria judaica ...... 38 Fig. 20 Late developmental stages of the bisexual flowers in Parietaria judaica ...... 39 Fig. 21 Floral developmental stages in Gesnouinia arborea ...... 40 Fig. 22 Several developmental stages of the female flowers in Soleirolia soleirolii ...... 41 Fig. 23 Floral diagrams of the representative species...... 41

1 Introduction Urticaceae is one of the largest wind-pollinated families in the order Rosales with ca. 50 genera and more than 2000 species. The family is most diverse in the Tropics, with the highest number of genera found in SE Asia, followed by Africa and Tropical America. A smaller number of temperate genera are common in their distribution (Woodland 1989; Friis 1992). With the exception of the tribe Cecropiaea, species-richness is highest above 500 masl and below 2000 masl, while the generic richness is highest at forest margins and disturbed habitats (Friis 1992). There are genera adapted to dry habitats (e.g. Forsskaolea, Parietara, Obetia) (Friis 1988,1989) or alpine vegetation (Urtica, some Elatostema and Pilea in New Guinea) (Friis 1992). The family is dominated by lignified taxa, from large terrestrial trees (in Dendrocnide, Cecropia, Musanga, Pourouma etc), to shrubs (Boehmeria, Pouzolzia, Phenax, etc.), shrublets (Parietaria, Pilea, Elatostematoides, Urtica), climbers (Poikilospermum, Urera, etc.) and succulent herbs (Elatostema, Procris, Pilea). They are rarely hemi-epiphytes (Coussapoa) (Berg et al,1990). The family is united by a combination of several derived character states including basal ovary, single orthotropous ovule, obtuse tipped conical hairs associated with the stigma, elastically inflexed stamens (except Cecropieae). There are four major tribes that are well supported and recognized at present: Boehmerieae, Elatostemeae, Urticeae, and Cecropieae (Wu et al. 2013; Treiber et al. 2016).

1.1 General morphology The flowers of Urticaceae species are usually small, greenish, unisexual, monoecious, dioecious or polygamous with 2-4 inconspicuous tepals or a tubular perianth. The male and female flowers in Urticaceae are highly dimorphic. In male flowers, stamens are always isomerous with tepals and antetepalous, and the inflexed stamens straighten at anthesis to eject the pollen grains are characteristic for the Urticaceae s.s (i.e. except Cecropieae) (Berg 1977). This type of explosive pollen release is a specially developed adaptation to anemophily, which occurs in all Urticaceae (except Cecropieae), the majority of the Moraceae, and Celtis laevigata Willd. of Ulmaceae (Berg 1989). Unlike the male flowers, the female flowers of the family are very heterogenous. The perianth consists of either free or connate or basal fused tepals, or even absent (e.g. Forsskaolea). The ovary consists of a single carpel and a basal, orthotropous ovule. The stigma is usually formed by clusters of trichomes with a long style or sessile. The basic forms of stigma shape are penicillate-capitate, elongated ligulate and 6 filiform, with various variations into subulate, peltate, circular, semilunar or spatulate in different cases (Friis 1992; Wu et al. 2015). The basic inflorescence form of the family is a compound cyme or panicle, either with a dichasial branching arrangement, or clusters of florets separated by parts of the stalk, so-called "interrupted spikes" (Friis 1992). The cymes can have all of their branches truncated (Boehmeria, Pouzolzia, Phenax, Rousselia), or truncated and fused (Elatostema), or only lateral branches truncated (Myriocarpa), or they can be regularly branched (Urera, Poikilospermum) or irregularly branched (Laportea, Urtica, Pilea). The “interrupted spikes” are frequently found among Boehmerieae, Elatostemeae and Urticeae, especially in Boehmeria and Myriocapa, but are absent in Cecropieae (Friis 1992). Cecropieae, on the other hand, have spikes in digitate clusters with florets condensed arranged and the whole inflorescence is normally enveloped by a caducous spathe (Berg 1977).

Separation of the four tribes morphologically is not easy. Generally, Urticeae is distinct by bearing stinging emergences, always has a rudimentary ovary in male flowers and two pairs of tepals in different size in female flowers. Elatostemeae is usually juicy and occurs in moist areas, lacking (or rarely having) pistillode in male flowers but with three scale-like staminodes that function in ejecting the achenes (Friis 1992). Boehmerieae possesses the highest level of diversity in female morphology and is characterized by a laminate rudimentary ovary in male flowers and a tubular perianth in female flowers. Bisexual flowers occur in Parietaria, Pouzoulia, and a few other genera within Boehmerieae. The former Forsskaoleae is special in the absence of female perianth, though a tubular perianth of somewhat uncertain nature occurs in Australina (Friis 1992). Cecropieae is unique in having compound leaves and an amplexicaulous stipule with a combination of characters like woody, strictly dioecious, bearing spikes or catkins and straight filaments.

1.2 Classification history Despise a long taxonomic history of Urticacae, it not easy to separate the family into tribes or subfamilies distinctly because many of them share a great amount of morphological variations between groups. The family was first divided into 6 groups (Elatostemeae, Urereae, Boehmerieae, Parietarieae, Forskahleae, and Cecropieae) (Gaudichaud 1830 in Friis 1989). H. A. Weddell (1854, 1856, 1869) published three works of the Urticaceae classification between 1850 to 1870 and recognized only 5 tribes -- Urticeae, Lecantheae (Procrideae), Boehmerieae, Parietarieae, and Forsskaoleae. Cecropieae was excluded in all of his studies, 7 because of its morphological similarities to Moraceae. And this was followed by Engler’s classification system and recognized by many people for a long time (In Engler & Prantl 1888). Based on a group of morphology, anatomy and cytology character distributions among the family, Friis (1989) maintained the main tribes of previous systems but with Boehmerieae, Parietarieae and Forsskaoleae grouped together into one tribe as Boehmerieae because they share the characters of a lanate or pilose pistillode in male flowers, tubular perianth mainly in female flowers, frequent arachnoid indumentum and punctiform cystoliths. The recent molecular work has agreed Friis’s opinion on grouping the former Forsskaoleae and Parietarieae together with Boehmerieae forming the largest complex in the family and indicates the Cecropia groups as the fourth clade within the family (Wu et al. 2013; Wu et al. 2015).

As the major controversial group in the family, Cecropieae has less synapomorphies with the Urticaceae s.s. than with Moraceae and thus possesses a more complicated classification history. The group was first mentioned by Dumortier (1829) including only Cecropia and Coussapoa within the Artocarpideae Dumortput. Engler (1989 in Treiber et al. 2016) grouped Cecropia, Coussapoa, Pourouma, Myrianthus, Musanga and Poikilospermum together as a subfamily of Moraceae, Conocephaloideae. Chew (1963) suggested to split the clade into the small-seeded group (Cecropia, Coussapoa, Poikilospermum, and Musanga) that should be replaced to Urticaceae and the big-seeded group (Myrianthus and Pourouma) which remains in Moraceae, in order to fit in the exist natural families. The whole clade was transferred into Urticaceae mainly based on the urticaceous characters, i.e. a simple stigma and an orthotropous, basal ovule (Corner 1962). However, Berg (1978) proposed a new family Cecropiaceae, considering its distinct differences from the Urticaceae, especially in habit and floral characters. But the agreement was hardly reached on the position of the clade, because of all these morphological and anatomical similarities they have among those groups. This was not solved until the molecular phylogenetic analysis revealed an inclusive genetic relationship between Urticaceae and Cecropia clade and thus four major clades have been proposed and acknowledged at present (Datwyler & Weiblen 2004; Zerega et al. 2005; Monro 2006; Hadiah et al. 2008; Clement & Weiblen 2009; Wu et al. 2013; Treiber et al. 2016). And the name Cecropieae Gaudich. has finally been validated in the recent review of Urticaceae nomenclature (Conn & Hadiah 2009).

1.3 Phylogeny and evolutionary importance of Urticaceae Although tremendous variation in both morphology and biogeography happens within the urticalean families with some confusing complexity changing systematic places now and then, Urticaceae together with Moraceae and Ulmaceae forming a well-supported monophyletic branch in Rosales now (Berg 1977, 1989; Savolainen et al. 2000a; Savolainen et al. 2000b) (Fig1). And the phylogenetic position of Urticaceae as the most derived family of the urticalean branch sister to Moraceae (Fig) is also confidently supported by molecular data (Sytsma et al. 2002; Soltis et al. 2011; Wu et al. 2013). The four tribes of Urticaceae are also well-supported by the molecular data (Hadiah et al. 2008; Wu et al. 2013; Kim et al. 2015), however morphologically, the classification can be rather disorganized due to the independent variation of characters between individuals (Wu et al. 2015), especially in the case of Cecropieae.

Fig. 1 Phylogeny of the Rosales. (Stevens 2015) 9

As the biggest wind-pollinated branch in Rosales, the urticalean lineage has raised a number of questions in the evolution of anemophilous flowers in terms of sexuality, floral reduction and merism, gynoecial modifications, inflorescence simplification and so on. Compared to the other Rosales families, the former Uritcales represents a progressive reduction of flowers present as unisexual, apetalous and pseudomonomerous as well as lacking a hypanthium, which is the main synapomorphies of the order (Ronse De Craene 2012). Apetaly is widespread within the order, but extremely concentrated in Urticales. Ronse De Crane (2003) proposed that the petals in these families have the same origin as stamens and arose through homeosis (a developmental process where an organ is transformed and replaced by a homologous part arising in a continuous series) from the outmost stamens and hypothesized that apetaly is the ancestral character for the Rosales with the petals regained occasionally during the evolution. However, this theory is under the consideration that the tepals of urticalean families are from a sepal-origin rather than a petal-origin and this is no direct proof in previous studies. Thus, floral developmental study is fundamental and crucial in clarifying the nature of the tepals and to contribute to the understanding of the perianth evolution in Rosales. Unisexuality is another main adaptation to wind-pollination and there are two type of ways to generate unisexual flowers, either by abortion of certain sexual part into nonfunctional rudiments or from inception without vestigial sexual organs (Mitchell & Diggle 2005). In Urticaceae, both cases are present but seem to have a different preference within different genders. Besides, bisexual flowers also occur in several genus of Boehmerieae. This has raised the question of the ancestral stage of the sexuality in Urticaceae, whether the small number of bisexual species is remnant of the ancestral bisexual flowers or a secondarily derived acquisition. Understanding those evolutionary trends in Urticaceae will definitely contribute to the understanding of the problematic evolution route of the Rosales.

Fig. 2 Phylogeny of the four major tribes of Urticaceae. 10

1.4 Economic importance Not all the Urticaceae species are economically valuable, but there are quite a few ones have been widely cultivated and used in various ways by locals communities, especially species in Urtica, Boehmeria, Pilea, Laportea, and Girardinia. The most common uses of the family are in textiles. The fibers of certain species are of high quality and thus can be made into cloth, ropes, fishing nets, amate paper and other industrial materials (Woodland 1989; Friis 1992; Heywood et al. 2007). Boehmeria nivea Gaudich and Laportea canadensis (L.) Wedd. are two major resources of fibers in Southeastern Asia (Hutchinson 1967; Woodland 1989; Friis et al. 2003; Heywood 2007). The product of B. nivea is commonly known as Ramie. In Central and Southeastern China, Girardinia diversifolia (Link) Friis is also widely cultivated to for the fiber named “Hong Huo Ma”, which is ideal for making ropes and is used in Traditional Chinese Medicine (TCM) for detoxication.

Some Urtica, Laportea, and Girardinia species are edible vegetables and the boiled young shoots can be cooked into fresh and delicious dishes (Friis et al. 2003). The seeds of the nettles are squeezed for cooking oil in some place, as comparable to the seeds of hemp, sunflower, and flax in protein and fat content. In Europe, Urtica is considered as abundant in carotene, chlorophyll, and Vitamins A, E, and K, and provided as an extra food supplement after extraction (Stern 1943 in Woodland 1989). Tribe Urticeae is well known for having stinging hairs that can cause persistent pains and paralysis, especially Urtica and Dendrocnide, and Urtica pollen is one of the main causes of hay fever (Heywood 2007). There are also species that have been used in medicine. Urtica dioica L., U. cannabina L., and U. urens L. have been reported as effective in rheumatism and arthritis treatment, as well as in the cure for snake bite in TCM. Many Urtica species are cultivated as pot, tea or medical herbs in Europe and North America (Woodland 1989).

Many species in the Elatostemeae are often cultivated as indoor or outdoor ornamentals because of their large and distinct leaves with variable shapes and colours as well as some other vegetative characters (Friis 1992; Heywood 2007). Pellionia repens (Lour.) Merr., Pilea cadierei Gagnep. & Guillaumin, P. microphylla (L.) Liebm., and P. peperomioides Diels are popular choices in Asia, Europe and other places around the world (Friis et al. 2003). Soleirolia soleirolii (Req.) Dandy is often used in greenhouses as a fast-growing carpeting ground-cover (Heywood et al. 2007). However, in farming and horticulture, the nettles are 11 always considered as troublesome weeds, which usually includes Urtica and Parietaria (Friis 1992).

1.5 Aim of study Despite the importance of pistillate floral morphology in generic delimitation, its great variation across the family and the existence of a robust phylogeny for the Urticaceae there has been no attempt to classify pistillate floral developmental and so establish hypotheses of homology for these key morphological characters. Doing so would provide a robust framework for studying floral evolution across the family. In addition there remain several basic elements of flora morphology that remain controversial or unevaluated: whether the tepals are of sepaloid or petaloid origin and whether bisexual flowers represent a derived or basal condition in the family and the function of highly conserved obtuse tipped conical hairs associated with the stigma. In the light of the above the specific aims of this study were: produce floral diagrams for at least one representative of each tribe in the family use floral development to test whether tepals are sepaloid, petaloid or neither in origin use floral development to test whether bisexual flowers are secondarily derived from bisexual inflorescences or a reversal to a hypothesized ancestral state use floral development to establish hypotheses for the origin of wind-pollination and its relationship with the female variation

2 Materials and Methods

2.1 Sample collection The samples used in this study were mainly collected from living materials at the Royal Botanic Garden, Kew, the Royal Botanic Garden Edinburgh, and the personal collections from Dr. Alexandre Monro. 11 out of ca. 50 genera with one species in each were involved in this study.

Materials were fixed immediately in FAA (70% ethanol : glacial acetic acid : formaldehyde=90:5:5) after collected and then stored in 70% ethanol. Both blooming flowers and buds were dissected under a ZEISS Stemi SV 6 stereo-microscope and preserved in 70% ethanol before going through the critical point drying and SEM scanning. Both spirited and pickled flowers were observed under a ZEISS Stemi 2000-C stereo-microscope and photographed by an AxioCam MRc 5 (ZEISS) digital camera.

Table 1. Species list of the material used in this study Species Name Collection No. Place of Collection Cecropia sp. JDS 2 Unknown Forsskaolea tenacissima L. Amt 2016 Spain Gesnouinia arborea (L. f.) Gaudich JD009 RBG, Kew Laportea aestuans (L.) Chew AM7604 Unknown Myriocarpa stipitata Benth. JD008 RBG, Kew Parietaria judaica L. JD010 RBG, Kew Pilea grandis Wedd. 19697471C RBGE Pouzolzia zeylanica (L.) Benn. AM7499 Unknown Rousselia humilis (Sw.) Urb. AM7463 Unknown Soleirolia soleirolia (Req.) Dandy. AM7641 Unknown Urtica dioica L. JD011 London

2.2 Critical point drying The dissected samples were transferred into the CPD carrying basket with six cylindric slots. Each species occupied a single slot each time and the label numbers on the basket with 13 the species name related were noted carefully. The samples were then dehydrated in an ethanol-acetone series:

70% ethanol ………………………………… 15min 95% ethanol ………………………………… 10min 100% ethanol ………………………………. 5 min 100% acetone ……………………………… 5min 100% acetone ……………………………… 5min

Once the samples were finished with this procedure, they were swiftly transferred into an Emitech K850 Critical point dryer and went through the process of critical point drying using liquid CO2 to displace acetone. The dryer was pre-cooled to around 4 ℃. Before the samples being heated to maximum 38℃, ten times exchanging of acetone to liquid CO2 was performed and the critical point drying was achieved when the temperature rose up to 35℃.

After the chamber was fully de-pressured while the temperature was maintained, the specimens were quickly removed to a desiccated container with dried silica gel.

2.3 Scanning electron microscope After the critical point drying, the specimens were mounted carefully on metal stubs using sticky carbon discs under a ZEISS Stemi SV 6 stereo-microscope and arranged in turn by species and genera. Each stub was labeled with a number using a diamond pen and recorded clearly. The stubs were then sputter coated with platinum inside an Emitech K575X Sputter Coater machine for conducting the electrons later on in the microscope. The procedure was repeated two to three times in order to let the samples fully coated and reduce the charging during the scanning.

Once the coating was finished, the samples were ready for scanning. A maxmum of 8 stubs were placed each time into a Leo Supra 55VP (Zeiss) Scanning Electron Microscope. Images were adjusted and taken under an acceleration voltage of 5kV (rarely 4.5kV) and a work distance around 10cm. The images were then edited, combined and labeled using Adobe Photoshop CC. 14

3 Results

3.1 Vairable morphology of the female flowers All the flowers examined showed a highly reduced pattern in different floral parts. Generally, the single flower is associated with a bract (and two bracteoles). The perianth is mostly tetramerous or tirmerous, rarely dimerous with free tepals or perianth fused in different degree. Flowers tend to be zygomorphic due to the tepal heteromorphy and/or gynoecium orientation. Most species observed have a trichomatic stigma in a capitate shape, rarely have an elongated penicillate stigma. The ovary often possesses a longitudinal grooved scar, especially in younger flowers. Some species have a short gynophore with or without a flattened base. Staminodes are only observed in P. grandis. Various types of trichomes are present among different species. A Summary of observed characters among the 11 species is given in the Appendix.

3.1.1 Urticeae Urtica dioica The individual plant of Urtica dioica can be either monoecious or dioecious, but the inflorescences are unisexual. The female inflorescences are paniculate, growing from the axil of the vegetative leaves with a lanceolate bract at the base (Fig. 3A). Each single flower is subsessile and surrounded by a small hispid bract (Fig. 3B). Mature flowers are green without clear differentiation of sepals and tepals. The four setulose tepals appear as two series: The outer two are larger and spreading while the inner two are much smaller and usually tightly surrounding the ovary (Fig. 3B, 12A, 12C). Unicellular trichomes with sharp tips and rough surfaces arise sparingly from the surface of bracts and tepals, as well as peduncles (Fig. 3B, 4B, 12A). The gynoecium is distinct with a transparent, tufted-capitate, brush-like stigma (Fig 4A, 4C). The multicellular trichomes arise right from the epidermal cells of the ovary and can grow up to about 0.4mm (Fig 4C, 4D). The surface of the ovary is smooth. Staminodes are absent.

Laportea aestuans The inflorescence of Laportea aestuans is unisexual, while the plant is monoecious. The female flower is monosymmetric with four heteromorphic tepals. The anterior tepal is rather large and funnel-shaped with a unicellular elongated trichome at the apex (Fig 4E, 4F). The other three are half-fused in young buds and become free in mature flowers (Fig 4E). The 15 lateral two usually can grow much larger than the posterior one (Fig 4F). Multicellular glandular trichomes are distributed along the margin of the tepals and sparsely on the tepal surfaces (Fig. 4E,4F). The gynoecium is oblique and slightly twisted at the style (Fig. 4F, 4G). The stigma is similar to that of U. dioica with multicellular trichomes (Fig. 4G). The ovule is long, (sub)basal, orthotropous, with outer integument a little shorter than the inner one (Fig. 4H).

Fig. 3 Morphological diversity in Urticaceae. A-B Urtica dioica; C Cecropia glaziovii; D-E Myriocarpa stipitata; F Soleirolia soleirolii; G Gesnouinia arborea; H-I Paritaria judaica.

Fig. 4 A Female flower taken off the outer tepals. B Detail of the tepal surface, showing trichomes. C Detail of the gynoecium. D Detail of the stigma. Note the multicellular stigmatic trichomes. E-H Laportea aestuans. E Part of the inflorescence. Note the conspicuous anterior tepal. F Lateral view of the female taken off a lateral tepal. G Detail of the stigma. H Longitudinal section of the ovary, showing a basal orthotropous ovule. Bars A, B=20μm; C, E, F=30μm; D, G=10μm; H=100μm. 17

3.1.2 Elatostemeae Myriocarpa stipitata Myriocarpa stipitata is monoecious with numerous pendulous male and female catkins hanging down from the axil of the petioles (Fig. 3D). In female inflorescences, all the sessile or subsessile florets are attached to one side of the peduncle, with a small lanceolate bract derived closely (Fig 3E, 5A). Unicellular spike-like trichomes with rough and wavy surfaces spread all over the peduncles. The structure of the female flower is quite simple, with only two tepals visible at maturity (Fig. 5B). According to the developmental study presented below, the two tepals are the anterior and posterior ones, in respect of the normal four in Urticaceae (Fig. 23C). The two tepals are slightly setulose and fused at base. No staminodes are observed and the gynoecium arises immediately from the receptacle without being lifted by a gynophore (Fig. 5C). The stigma is spatulate with the elongated side opposite the posterior tepal and a short style beneath (Fig. 5B, 5C). The trichomes on the stigma are multicellular and spreading into all directions (Fig. 5D). The surface of the gynoecium is rough and with hooked unicellular trichomes sparsely distributed, and no groove is found on the ovary in matured flowers. There are one carpel and a single orthotropous ovule basally attached to the placenta. The ovule is enclosed by two integuments, the tips of which are modified into hair-like structures, especially the inner integument (Fig. 15C, 15D).

Pilea grandis Pilea is a genus without stinging emergences and hardly pubescent or hispid. Pilea grandis is monoecious but the inflorescence is unisexual. Each pistillate flower is petiolate or subsessile and surrounded by a small bract and two bracteoles at the petiole base (Fig 5E). A secondary floret may develop at the axil of the bracteoles (Fig 5E, 5F). The females are strongly zygomorphic. There are only three tepals in mature female flowers, of which the posterior one is distinctly larger and usually 3-toothed (Fig 5E-G). At the inner base of each tepal, there is a staminode with the size related to the tepal size (Fig 5H, 5I). The stigma is sessile with numerous unicellular finger-shaped tentacles forming a capitulum (Fig 5E-H). The ovary is about 0.1-0.15mm and glabrous at the ovary surface. A shallow groove caused by carpel folding can be more or less observed on the ovary opposite the posterior tepal in mature flowers (Fig 5H). A gynophore is absent in this species. 18

Fig. 5 Female variation in Elatostemeae. A-D Myriocarpa stipitata. A Detail of part of the inflorescence. B Lateral view of the female flower. C Lateral view of the female flower taken off half of the perianth. D Detail of the stigma. E-I Pilea grandis. E Part of the inflorescence. F An inflorescence unit. Note the free lateral tepals at maturity. G Lateral view of a female flower showing the heteromorphism of the tepals. H Apical view of the female flower without the anterior tepal. Note the position of the staminode. I Detail of the staminode. Bars =100μm; D, H=30μm; I=10μm.

3.1.3 Boehmerieae Parietaria judaica Parietaria is one of the genera that have both unisexual and bisexual flowers. The female flower of Parietaria judaica always appears together with bisexual flowers on the same inflorescence (Fig. 3H, 3I), which is termed as gynomonoecious (with both female and bisexual flowers on the same individual). The flowers clustered densely forming decussate compound cymes at leaf axil (Fig 3I, 6A). Usually only one female flower grows at the middle of the inflorescence cluster, as the first one to develop (Fig 3I). Each floret is surrounded by a bract and two bracteoles. Both female and bisexual flowers are tetramerous with four greenish pubescent tepals merely fused at base. In female flowers, only a gynoecium is lifted by a short fleshy gynophore inside the perianth, with no evidence of staminodes. There is a slim style with a red brush-like capitate stigma sticking out of the clingy perianth, which is easily broken at maturity (Fig 3H). In bisexual flowers, four stamens are inflexed and surrounding a similar gynoecium to the female ones (Fig. 3I, 6A, 6B). A gynoecium with a similar style and stigma reaches out beyond the perianth before the anthesis, or specifically, the male stage, and drops easily after fertilization (Fig 3H, 6C). There is a more or less distinct groove on one side of the ovary slightly underneath the style. The smooth ovary consists of a single carpel with a basal orthotropous bitegmic ovule. The top of the outer integument is often modified into trichomatic structures when matured (Fig 20F). Trichomes are dense both on the tepal surface and the receptacle (Fig 6B, 6C).

Gesnouinia arborea Gesnouinia is much more similar to Parietaria in the arrangement of the inflorescence (Fig. 3G). The female flower usually grows at the center of an inflorescence unit (Fig. 21A, 21B). But the bisexual flowers in this case is more precise to be called functionally male flower because the gynoecia in these flowers are pistillodes. Here in Gesnouinia arborea, both the male and female flowers are mostly tetramerous but occasionally trimerous, even on the same inflorescence (Fig. 21B). The stigma of the female flowers is also brush-like but much more elongated, with at least two types of hairs and is only densely hirsute on one side; a distinct style can be found as well (Fig 6F-H). A short fleshy gynophore (Fig. 6G) and an unsealed groove on the ovary can be found in G. arborea, as in P. judaica. In the male flowers, the gynoecium structure is also quite well-developed with a long style, but without any sign of an initiation of the trichomatic stigma (Fig. 6E). Only several unicellular rough hairs appear sparsely on the style. 20

Soleirolia soleirolii Soleirolia is a monotypic genus with only one monoecious species Soleirolia soleirolii. The flowers are usually solitary at leaf axil (Fig. 3F). Each flower is encompassed by an involucre of three segments derived from a same primordium (Fig. 7E, 22A). In female flowers, the perianth is basically tubular, with three or rarely four tepal segments hairy at the margins (Fig. 7A, 7C). Capitate glandular trichomes are frequently distributed on the surface of both bracts and tepals (Fig. 7C). The gynoecium is lifted by a gynophore and the stigma is a brush-like capitulum formed by a cluster of finger-shaped unicellular glabrous tentacles (Fig 7B). The male flower is tetramerous with a conspicuous pistillode (Fig 7E, 7F). The ovary groove can be seen on the pistillode of male flowers (Fig 7E), but not observed in female flowers due to the limitation of mature flower materials.

Rousselia humilis Rousselia humilis is a monoecious species similar to Soleirolia soleirolii in male flowers, but very different in female ones. Female flowers are growing in pairs and each is surrounded by a big bract possessing capitate glandular trichomes on the surface and hooked spines at the margin (Fig A). The perianth is adnate to the ovary with two small hairy segments at the top, surrounding the short, slim style (Fig 8B, 8C). The conspicuous stigma stretching out beyond the perianth tube is formed by numerous finger-shaped unicellular smooth trichomes (Fig 8D). The ovule is heart-shaped at maturity and the micropyle tends to be tentacular (Fig 8C). No gynophore or staminode was observed in this species. There is a leafy appendage between the paired florets with numerous long, slender, and rough trichomes at the margin (Fig 8C), of which the origin and function are still unknown.

Fig. 6 Female variation in Boehmerieae (1). A-D Parietaria judaica. A Apical view of part of the inflorescence unit. B Detial of a bisexual flower showing the gynoecium with functional stigmatic trichomes. C Lateral view of a female flower taken off two tepals. D Detail of the stigma. Note the protective hairs surrounding the stigmatic trichomes. E-H Gesnouinia arborea. E. Apical view of a functionally male flower. Note the conspicuous pistillode. F Lateral view of a female flower with a tubular perianth. G Lateral view of a female flower with half of the perianth. The arrow indicates the gynophore. H Detail of the functional stigma in female flowers. Bars A,B,C=100μm; B,F,G,H =20μm; D,E=10μm. *Figure abbreviations: B=Bract; Bt=Bracteole; P=Perianth; T=Tepal; S=Stamen; G=Gynoecium; Sti=Stigma; F=Female; M=Male; Fl=Floret. 22

Fig. 7 Female variation in Boehmerieae (2). A-F Soleirolia soleirolii. A Apical view of the female flower showing the 3-toothed perianth. B Detail of the gynoecium. The arrow indicates the gynophore. C Lateral image of the female flower, showing the bowl-shaped bract and bracteoles and the fused perianth. D Detail of the stigma. E Apical view of the male flower. F Lateral view of the male flower. Note the conspicuous pistiollode with an aborted stigma. G-I Fosskaolea tenacissima. G Apical view of the inflorescence. H Detail of the female flower. The arrow indicates the gynophore. I. Longitudinal section of the ovary showing the basal ovule. Bars=100 μm; A=20 μm; B,C=10 μm; H=30.

Fig. 8 Female variation in Boehmerieae (3). A-D Rousselia humilis. A Lateral view of a female inflorescence unit. Note the large bract surrounding the flower. B Lateral view of a single female flower. Note the tubular perianth and the brush-like stigma reaching out. C Longitudinal section of the ovary showing the ovule. D Detail of the stigma. E-F Pouzolzia zeylanica. E Lateral view of the female flower. F Apical view of the female flower with position of the gynoecium. Note that the perianth is tightly adnate to the ovary. Bars =100μm; B=30μm; D=20μm.

3.1.4 Cecropieae Cecropia sp. Cecropia is a distinctive genus of dioecious trees in the Neotropics. Both male and female flowers are in compact spikes enclosed by a spathe before anthesis (Fig 3C). The species of the Cecropia sample studied here is unidentified yet. The female flowers are densely packed on the spike and padded in a white cotton-like indumentums. Each flower consists of a tubular perianth and a typical urticaceous gynoecium (Fig 9A-C). The perianth is covered by a mass of threadlike hairs and some eglandular marginal trichomes (Fig 9A). The stigma is brush-like with a tuft of smooth multicellular tentacles obliquely developed and connecting to a longitudinal groove running to the middle of the ovary with postgenital fusion at some points (Fig 9C). However, it is uncertain whether this groove will completely fuse and disappear at maturity until older inflorescences are observed. Spine-like trichomes are present occasionally on the short style (Fig 9C, 9D). The ovule is immature here and shows the faster development of the inner integument than the outer and a tendency of a trichomatic micropyle as mentioned in other species before (Fig. E).

Fig. 9 Female variation in Cecropieae (Cecropia sp.). A Lateral view of the female flower showing the tubular perianth and the cotton-like indumentum. B. Longitudinal section of the perianth showing the position of the gynoecium. The arrow indicates the gynophore. C Lateral view of the gynoecium. The arrow indicates the groove. D Longitudinal section of the ovary showing the basal ovule. E Detail of the ovule. Bars A,D=30μm; B=100μm; C,E=20μm.

3.2 Floral development observation of different tribes Five out of the ten species discussed above were dissected from young to old inflorescences and scanned with SEM, in order to reveal the floral development process of Urticaceae. This covers three of the four recognized Urticaceae clades: Urticeae, Elatostemeae, and Boehmerieae. The spikes of Cecropia species seem to be synchronously developed, thus a series of immature inflorescence will be needed to do the developmental study, which is unpractical this time.

3.2.1 Urticeae Urtica dioica The inflorescence of U. dioica is an indeterminate and compound raceme, and those female flowers arise closely to each other, forming compact units. Thus, it is difficult to differentiate the bract for each floret. But generally, the primary bracts or involucels of the units form first and a floral primordium is soon formed in the axil of the bract (Fig.10A). The initiation of the four tepals is sequential. The first tepal is initiated at the left or right side of the primordium referring to the axis and grows fast, followed by the second one arising in the opposite position (Fig 10B-F). Two inner tepals are initiated when the first one has already overtopped the flower and appear to be more synchronous (Fig 10G, 10H). While the perianth is forming, an elliptical gynoecium primordium protrudes at the middle, and becomes more distinct and round during the inner tepal formation (Fig 10D-H).

At the time when the inner tepals are well differentiated, the gynoecium primordium is differentiated into a single basal arising ovule and the ovary wall (Fig. 11A). However, stages of early ovule initiation were not available. Then the ovary wall starts to grow upwards with one side of the primordium, usually opposite the first tepal, developing much faster than the other side, forming an oblique cylinder (Fig. 11B, 11C). Because of this unequal development at the beginning, the ovule is enclosed by the ovary from one side only leaving an open point along the other side (Fig. 11D-F). The trichomatic stigma begins to develop from the epidermis of the ovary apex before the carpel is completely sealed (Fig. 11E, 11F). Those uniserial epidermal cells enlarge and elongate from the faster-developed side towards the groove, and divide into multicellular trichomes at a length of about 0.4mm (Fig. 12A-C). The gap on the ovary wall eventually disappears at maturity. In the ovule, the integuments develop slower than the nucellus (Fig 12D), and the outer integument is even still shorter than the 26 inner integument (Fig 12E). There seems to be a tendency of the outer integument to produce trichomes surrounding the micropyle, but no distinct modifications were seen in my samples.

Fig. 10 Perianth initiation stages in Urtica dioica. A Apical view of the position of a floral primordium. B Initiation of the first tepal. C Apical view of an early primordium with two outer tepals initiated. D Frontal view of an early primordium showing the initiation of the globular gynoecium primordium. E. Frontal view of an early primordium showing the faster grouth of the first tepal. F. Apical view of an early primordium. G Lateral view of an early primordium showing the formation of two inner tepals. H Frontal view of the flower with differentiated outer tepals. Bars=10μm.

Fig. 11 Gynoecium developmental stages in Urtica dioica (1). A Early stage in the carpel formation. Note the differentiation of the ovule and the ovary wall. B Initiation of the unequal development of the ovary wall. C Lateral view of a young buds showing the unequal development of the ovary wall. D Apical view of a gynoecium at later stage. Note that the ovary wall encloses the ovule from one side. E Apical view of a young bud showing the initiation of the stigma. F Apical view of a young bud showing the ovary groove before the carpel sealed. Bars=10 μm; C=20 μm. 28

Fig. 12 Gynoecium developmental stages in U. dioica (2). A Frontal view of an young inflorescence. Note the oblique developmental sequence of the stigma and the differentiation of the inner tepals. B Lateral view of a young female flower with two inner tepals. C Apical view of a mature female flower. Note the tread-like stigmatic trichomes and the decussate- dimerous tepals. D Early developmental stage of the ovule development. E Later developmental stage of the ovule. Bars=20 μm; A=30 μm; D=100μm.

3.2.2 Elatostemeae Myriocarpa stipitata The female flowers at different developmental stages grow densely on the pendulous catkins covered with spine-like trichomes (Fig 3E, 5A). This somehow made the observation of young flower buds very difficult, especially the identification of the bracts. Due to the limitation of time and material, the developmental process of M. stipitata is still a little confusing in both the perianth and gynoecium stage. The primordium is formed directly on the peduncle next to the bract and two tepals are first to differentiate while the center protrudes as a gynoecium primordium (Fig. 13A-B). One of the two tepals usually grows faster than the other (Fig. 13C-D). It is still a mystery whether the other two tepals are initiated or not, because in some images the perianth appears to be four-parted (Fig. 13E-F) while in others only two tepals are observed (Fig. 13G-H).

During the differentiation of the perianth, the gynoecium primordium invaginates at the middle forming an urceolate ovary and the ovule is derived inside (Fig. 14A-C). Then one side of the apical meristem of the ovary grows faster than the other, and either develops into a cover on the top of the hole (Fig. 14D) or gradually narrows the opening by the unequal division (Fig. 14E). Meanwhile, the epidermal cells at the apex begin to enlarge and dividing, as an initial of the brush-like stigma (Fig. 14F, 15A-B). While the multicellular stigmatic trichomes are developing, the basal, orthotropous ovule is also forming. The bitegmic ovule of M. stipitata looks quite special and seems to have the most complicated modification of the integument among all the genera examined. The inner integument grows much longer than the outer one and the apical cells of the integuments are divided and modified into trichomatic structures, especially the inner integument (Fig. 15C-D). Trichomes can be as long as 0.2mm.

Pilea grandis The bract and bracteoles develop first from the peduncular tissue and the floral primordium is formed under the protection of the two bracteoles (Fig. 16A). The early stage of perianth derivation is obscure due to the limitation of the samples, but a synchronous development of the whole perianth as a tube can be seen in some stages (Fig. 16B). The margin of the perianth then starts to split, first into one broad, rounded and two narrow, acute segments (Fig. 16C). Next the broad tooth splits again into two acute teeth and a flattened part in between (Fig. 16D). Eventually, the three segments break up into three free tepals (Fig. 16E). 30

Fig. 13 Perianth developmental stages in Myriocarpa stipitata. A Early stages of a female primordium with two tepals initiated. B Early stages in tepal initiation with one tepal taken off. Note the globular gynoecium primordium. C Perianth differentiated stage showing the unequal development of the two tepals. D Perianth differentiation stage. The gynoecium has begun to differentiate at this stage. Note the expanding growth from the base of the left tepal. E Apical view of a young bud showing the occurrence of two additional tepals (with stars). F Apical view of a young bud showing the associated bract and the additional tepal (with a star). G Apical view of a young bud showing the two differentiated tepals. H Apical view of a young bud showing the two tepals developed into different sizes. Bars = 10μm.

Fig. 14 Gynoecium developmental stages in Myriocarpa stipitata (1). A Initiation of the ovule. B Detail of a young unsealed carpel. C Lateral view of a young unsealed carpel within the perianth. D Apical view of a young bud with carpel begins to seal. E Frontal view of a young gynoucium showing the unsealed groove. F Initiation of the stigma from the epidermis of the ovary wall. Bars=10 μm.

Fig. 15 Gynoecium developmental stages in Myriocarpa stipitata (2). A Apical view of a young female flower showing the differentiation of the stigma. Note that an additional tepal present (with star). B Lateral view of a floral bud showing a young gynoecium with well- developed trichomes. C Longitudinal section of the ovary showing an early stage of the ovule. D Longitudinal section of the ovary showing a later stage of the ovule. Note the hairy modification of the inner integument. Bars= 20μm; A=10μm.

Fig. 16 Perianth developmental stages in Pilea grandis. A Apical view of part of the inflorescence showing the position of the bract and bracteoles. The floral primordium is covered under the bracteoles. B Early stages of perianth formation showing the congenital initiation of the perianth. C Lateral view of a young flower bud showing congenital perianth with an undefferentiated gynoecium. D Apical view of a young bud showing the unequal development of the perianth segments. E Late stage in the perianth development showing free tepals. Bars=10μm; A=30μm.

While the perianth margin is splitting, the gynoecium primordium gradually emerges from the inside of the perianth cup (Fig. 16B-E). However, the next stage of gynoecium development was not captured, until a pear-shaped unsealed carpel has formed (Fig. 17A-B). While the open-slot is narrowing, the apical epidermal cells begin to inflate and elongate, forming the smooth, finger-like, unicellular stigmatic trichomes in the end (Fig. 17C-F, 18A- C). The eglandular staminodes have already formed when the stigma formation initiates (Fig. 17D). The single ovule initiates at the base of the ovary and is orthotropous; the outer integument grows much slower (Fig. 18C), but encloses the inner integument and the nucellus at maturity (Fig. 18D).

Fig. 17 Gynoecium developmental stages in Pilea grandis (1). A Apical view of a young flower with an unsealed carpel. B Frontal view of the uncealed ovary. C Frontal view of a young bud showing the fusion of the ovary groove and initiation of the stigma. D Lateral view of a gynoecium in late stage. E Detial of the stigma formation in late stage. F Lateral view of a young flower. Bars=10μm. 35

Fig. 18 Gynoecium developmental stages in Pilea grandis (2). A Frontal view of an older flower bud showing the fusion of the ovary groove. B Some young flowers showing differentces of the stigmatic trichomes of different stages. C Longitudinal section of the ovary showing an early stage of the ovule. D Longitudinal section of the ovary showing a later stage of the ovule. Bars A,B=10μm; C,D=20μm.

3.2.3 Boehmerieae Gesnouinia arborea & Parietaria judaica Gesnouinia and Parietaria tent to follow a strongly regular and similar developmental process. Because the terminal female flowers are the first ones to develop in an inflorescence unit, it is much harder to get young materials for the floral development study. Only the development of the male or bisexual flowers is discussed here.

The inflorescence unit of Gesnouinia arborea is delimited by the involucre derived all from the same rachis (Fig. 21B). The female flower usually initiates in the middle, surrounded by a bract and two lateral bracteoles, and later the bracteoles become the bracts of the first two bisexual flowers (Fig. 21A-B). The male flowers can be either tetramerous (Fig. 6E, 21B, 21F-H) or trimerous (Fig. 21B-E). The earliest stage recorded in this study already has differentiated tepals, stamens, and gynoecium primordium, so it is unlikely to prove the initiation sequence of different organs. However, judging from the size of the primordial within a whorl, it is possible that the sequence of tepal initiation is spiral, as well as the stamens (Fig. 21B-F). Yet, the initiation sequence of the stamens may be different from the tepals even though they are isomeric because the largest stamen primordium is usually opposite the largest tepal (Fig. 21B-E). The development of gynoecium is also poorly understood. The gynoecial primordium differentiates from the globular primordium into an oblate globoid structure (Fig.21A-B) and grows into an ovoid shape later on (Fig. 21B). The ovule is initiated at this stage, as the alternitepalous side begins to protrude at certain point and sinks peripherally (Fig. 21C-F). After the ovule is formed, the margin of the ovary wall starts to fuse, leaving a distinct groove throughout the style and the ovary during the process, and restricts the crevice only to the ovary in the end (Fig. 21G-H).

The inflorescence of Parietaria judaica is very similar to that of Gesnouinia arborea, as well as the early developmental stages of perianth, stamen and gynoecium primordia differentiation (Fig. 19A-D). However, the initiation of the ovule seems a little different in Gesnouinia. Instead of the ovary surface protruding to form the ovule shape, in P. judaica the ovary wall invaginates peripherally and shapes the ovule inside (Fig. 19E-F). Meanwhile, the apex of the primordium is elongated to form the style (Fig. 19F). Later, the ovary margin fuses to enclose the ovule and leave a groove along the ovary (Fig. 20A-C). While the ovary wall is growing, the epidermal cells at the top of the gynoecium primordium also begin to divide and elongate, forming finger-shaped unicellular smooth trichomes from the apex 37 downwards, known as the stigma (Fig. 20A, 20D). The ovule starts to differentiate into a nucellus and two integuments after being completely enclosed by the ovary wall. The outer integument grows as long as the inner one, and the top margin is modified into short trichomes (Fig. 20E-F).

Fig. 19 Early developmental stages of bisexual flower in Parietaria judaica. A Apical view of a floral bud with primordia of tepals, stamens and gynoecium in an early stage. B Apical view of a young bud with tepals grow overtop the flower. C Apical view of a young bud with one stamen undeveloped. D Lateral view of a young bud with the gynoecium begins to differentiate at the apex. E Apical view of an inflorescence showing the ovule initiation in the middle flower. F Apical view of an unsealed carpel with one side elongated into a style. Bars=10μm; A,E=20μm.

Fig. 20 Late developmental stages of the bisexual flowers in Parietaria judaica. A Longitudinal section of a late flower bud. Note the ovary groove in front. B Lateral view of a near-mature flower bud without perianth. C Apical view of a near-mature flower bud showing the conspicuous ovary groove. D Detail of the stigma of bisexual flowers. E Detail of an immature ovule. E Lateral view of a mature ovule with hairy modifications at the integument apex. Bars A,C=30μm; B=20μm; D,E=10μm; F=100μm.

Fig. 21 Floral developmental stages in Gesnouinia arborea. A Part of the inflorescence showing the early floral primordium. B Apical view of a primary inflorescence unit with a female bud at the middle and two male buds on side. C Apical view of a trimerous male bud with differentiated perianth, stamen and gynoecium primordia. Note the ovule initiation from the ovary surface. D Apical view of a trimerous male bud. E Apical view of a trimerous male bud with the gynoecium more developed and the style/stigma starting to differentiate. F Lateral view of a tetramerous male bud showing the more developed stamen and ovary primordium. G Half flower of a young male bud showing the long pistillode groove and the aborted stigma. H Apical view of a near-mature male flower showing the conspicucous groove on the pistillode and the flattened gynophore. All bars=10μm.

Fig. 22 Several developmental stages of the female flowers in Soleirolia soleirolii. A Apical view of a trimerous bud. Note the undifferentiated and fused bract and bracteoles. B Apical view of a tetramerous bud with four developed tepals developed in sequence. C Lateral view of a tetramerous bud with an unsealed young gynoecium. Bars A=10μm; B,C=20μm.

Fig. 23 Floral diagrams of the representative species. 42

4 Discussion

4.1 Nature of perianth and meristic variation The perianth of male or bisexual flowers is mostly tetra- or pentamerous and morphologically homogeneous within the family, while the female perianth is more variable in number, size and texture (Friis 1989, 1992). Only the female perianth is discussed here. The nature of the perianth in Urticaceae has been generally believed to have a sepal-origin ( Bechtel 1921; Sattler 1973) but no direct evidence was presented in previous publications. Here, the developmental process of those species has given a positive support for this view. First, a distinction is needed here between a primary undifferentiated (tepals without differentiation between perianth parts) and a secondary undifferentiated perianth (petals are lost during evolution) (Ronse De Craene 2012). In Urticaceae, the tepals are basically greenish, hairy and always broad at base, sometimes not well-differentiated from the involucre. Occassionally they are even keeled, for example in Laportea (Fig. 4E-F), which normally only happens on the calyx in other eudicots. Most of the species have persistent perianth that can be observed in fruits (Friis 1989). Besides, although in some observed cases here, the perianth part initiates as a cup-shaped structure or within the same whorl, as observed in L. aestuans and P. grandis, the final positions of the tepals are always in different series at maturity. The initiation sequence of those tepals is not always strictly spiral as it should be in sepals, but is sequential, either alternately (e.g. Urtica), or rotationally (e.g. Soleirolia), or even more complicated (e.g. Myriocarpa) and the initiation and differentiation of the perianth is always faster compared to the gynoecium development. Thus, it is fair to say that Urticaceae has a secondary undifferentiated perianth.

Among the ten samples studied, at least four types of merism have been found. There are both conformities and variations within the tribes. The Urticeae tend to have a free, tetramerous perianth (Friis 1989), as observed in U. dioica and L. aestuans here. But they also show great differences in perianth appearance. U. dioica, because of the two series of tepals of unequal size, is disymmetric (Fig. 23A). In some treatments of Urtica (Woodland 1982; Boufford 1992; Friis et al. 2003), the outer two tepals are said to be smaller than the inner ones, including U. dioica. However, with reference to the development of U. dioica, it is obvious that the outer tepals arise much earlier and grow faster than the inner tepals (Fig. 11F, 12A), and they are not distinctly different in size in mature flowers in my observation. While the four tepals in U. dioica are comparable in appearance, those in L. aestuans are 43 distinctively different as one of the anterior ones is rather larger and better-modified in shape, which results in the decidedly zygomorphy of L. aestuans (Fig. 23B). This is also reflected in the stage of early perianth development (Fig 4E), when the anterior tepal has already differentiated but the other three are still small and mostly fused. This suggested that the posterior and two lateral tepals might initiate alomost synchronically but much later than the anterior one. The anatomical study has also revealed that no vascular strand is shared by both the anterior tepal and posterior tepal (Bechtel 1921). Elatostemeae is reported to have a great amount of variations in perianth, being either penta- to trimerous or very reduced to absent (Friis 1989). The two species, M. stipitata and P. grandis, have a dimerous and trimerous perianth respectively (Fig. 23C). In M. stipitata, a third (and a fourth) tepal primordium occasionally present in the early stage (Fig. 13E-F), yet their initiation is not clearly revealed. My hypothesis is that it is derived from the base of the first developed tepal as an appendage and failed to develop in later stages, thus becomes reduced or fused with the existing tepals eventually. It is also possible that the additional segment is derived from the deeply toothed tepal where the bases of the segments are still fused. The perianth formation of P. grandis is much clear as the three tepals are initiated together as a tube and become differentiated and separated in late development, which resembles L. aestuans to some extent. Merism of the Parietaria lineage (Soleirolia-Gesnouinia-Parietaria) is notable. Although all the female samples observed in G.arborea and P. judaica are tetramerous, there are trimerous male flowers in G. arborea and both tetramerous and trimerous flowers can appear on the same inflorescence unit. As for Soleirolia, the common type of female perianth is a 4-lobed tube (Weddell 1854; Weddell 1856; Friis 1992) (Fig. 7C, 22B), whereas some trimerous females are also observed (Fig 7A, 22A). However, I tend to believe that the trimerous flowers are productions of freaky development of the third and fourth tepals due to the space competition. Because the tepals of S. soleirolii are initiated sequentially in one direction, and the later two are always pressed towards the axis. Besides, unlike P. judaica and G. arborea, the bract and the bracteoles of S. soleirolii are derived on the same whorl and not distinctly differentiated from each other, which may indicate a primitive position of Soleirolia (Fig. 23F). Rousselia possesses a tubular perianth tightly clinging to the ovary wall and shallowly 2-toothed (Fig. 8A-D), which is very similar to members of its sister group Pouzoulia (Fig. 8E-F). The Cecropia species also has a tubular perianth, but because it is covered densely by hairs, it is hard to differentiate whether the tube is toothed at the top or not (Fig. 23G).

The reason for the changes of floral merism is always related to the space distribution within a flower and always controlled by specific developmental patterns (Ronse De Craene 2016). The predominant type of merism in the core eudicots is pentamerous (Doyle & Endress 2011; Ronse De Craene 2012, 2016) whereas in Urticaceae, the most commonly observed type is tetramerous, especially in male flowers. This can be a result of the compact cymose inflorescence and a reduction of the size for individual flowers. Besides, the great variation of merism from tetramerous to trimerous or dimerous could also be a consequence of the apetalous character, since the loss of any perianth parts will lead to a lower meristic stablility (Ronse De Craene 2016). The formation of the two whorls of tepals in Urtica can also be explained by the space restriction, where the outer two tepals show a faster growth in the early developmental stage and occupy much more space within the whorl.

4.2 Significant gynoecium characters Stigma and style The stigma form of Urticaceae is rather variable. The general morphology of the stigma is a cluster of unicellular or multicellular trichomes, but can be capitate, penicillate, ligulate, peltate, subulate, oblong, semilunar or spatulate in shape (Wu et al. 2015). The female flower of G. arborea has an elongated penicillate stigma (Fig. 6G-H), which is very similar to the female stigma of P. zeylanica (Fig. 8E-F). It seems that this type of elongated brush-like stigma occurs more frequently within Boehmerieae. The semilunar stigma of M. stipitata is a unique character of the genus (Wu et al. 2015). The trichomes seem to grow on an obviously oblique, flattened region, leading the whole flower to a zygomorphic pattern (Fig. 5B, 5D). Cecropia tends to have a peltate stigma that extents to the ovary groove (Fig. 9C). This character has independently evolved several times within the family and is considered as a synapomorphy of Cecropia and Oreocnide (Wu et al. 2015). Other species all possess a generally capitate trichomatic stigma. Among all the species observed, only U. dioica, L. aestuans and Cecropia sp. have slim, multicellular stigmatic trichomes, while all the others are unicellular and much more blunter at the tips. The two species of Urtica, as well as P. grandis, have almost no styles.

Gynophore A gynophore lifting the ovary up from the receptacle occurs in many species of Boehmerieae and Cecropia in this study. In the bisexual flower of P. judaica and the 45 functionally male flower of G. arborea, the receptacle is also swollen and flattened under the gynophore (Fig. 20A, 21H). The presence of a gynophore may have something to do with the inflorescence formation and sexuality of the flowers because it appears frequently in the clades where bisexual flowers are present. There is a possibility that the gynophore indicates a solitary flower ancestral to the current inflorescence, or the formation of bisexual flowers from the reduction of compound bisexual inflorescences.

Pseudomonomerous ovary All the species observed in this study only have a superior ovary with one carpel and an orthotropous ovule (sub)basally attached, as well as a single stigma/style. With reference to the primitive character state of the former Urticales, which is bicarpellate, this type of unicarpellate gynoecium should be considered as pseudomonomerous (Bechtel 1921; Berg 1989). However, there is no trace in the floral development showing any residue of a second carpel. The gynoecium developmental process observed here is compatible with the ontogenetic study of some Urtica and Parietaria by Cramer and Swann (Cramer & Swann 1966). The gynoecium formation process is strongly similar among different groups. It initiates as a globular primordium when the tepals are well differentiated or at least forming an obvious perianth tube. While one side of the primordium is growing faster than the other, the ovule emerges from an axile position by either sinking or protruding or a combination of both. The other side of the ovary primordium usually develops very slowly or remains undeveloped until the fast-growing side has gradually enclosed the ovule, leaving a groove due to the folding of the carpel (e.g. Fig. 17B, 19F, 21H). The shape of the groove varies among different species. For example, in P. grandis it is short and shallow while in female G. arborea, it finally becomes a small slit in maturity. It seems that all the species show at least a stage of bearing such a groove, however, whether the trace will be completely fused and vanished in older stages of maturity is still uncertain, because the limitation of the material. Only U. dioica, L. aestuans and M. stipitata have been observed with no sign of an unsealed ovary in mature flowers.

Ovule The single ovule of all the species is basal, orthotropous and bitegmic. The development of the ovule follows the common progress, where the nucellus arised first and progressively covered by both integuments. Yet in some species, the outer integument is much shorter than the inner one even in mature flowers, like L. aestuans and M. stipitata (Fig. 4H, 15D). In 46 other cases, the outer integument would grow as long as the inner at maturity and both enclose the nucellus inside as nornal, like P. judaica, U.dioica and F. tenacissima (Fig. 7I, 12E, 20F). However, the maturation time of the ovule seems different from that of the stigma, i.e. the stage when the stigma is functionally receptive and the linkage between the two needs further investigation.

Moreover, there seems to be a tendency for many species to have ‘tentacles’ around the micropyle, i.e. hair-like structures derived from the epidermal cells at the top of the integuments. Among the species where the ovule has been observed, M. stipitata has the strangest ovule, where both integuments are tentacular with the outer one bearing extremely long and interlaced hairs. P. judaica and F. tenacissima both have outer integument ‘tentacles’. In L. aestuans, U. dioica, and P. grandis, the cells around the micropyle tend to elongate and are segmented, but no real trichomes have shaped. In immature flowers of Cecropia, the inner integument has a higher tendency to form tentacular micropyle (Fig. 9D- E). The function of the integument hairs is unclear and there is no evidence in the morphological or developmental results here showing any direct connection or interaction between the ovule hairs and the stigmatic trichomes. My guess is that the hairs may play an important role in pollen transition and recognition if the pollen tubes are growing into the nucellus through the micropyle. However, to proof that, further anatomy and chemical test need to be done at pollination and fertilization stage.

4.3 Trichomes of Urticaceae flowers Most genera of Urticaceae have hairy flowers and the morphology of the trichomes is rather abundant. The trichomes in Urticaceae can be first divided into two types: stigmatic trichomes, which is specially functioned in the reproductive process, and protective hairs, which spread to different organs and are assumed to function in protecting the reproductive structures. For the stigmatic trichomes, there are generally two types. One is multicellular and whip-like, as possessed by U. dioica, L. aestuans and M. stipitata. The other type is unicellular and mostly finger-liked, as possessed by the other species such as P. grandis, S. soleirolii and Cecropia. The trichomatic stigma is preferable in pollen capture for wind- pollinated plants.

As for the protective hairs, classification mainly follows Gangadhara and Inamdar’s summary (1977)on the trichome types of “Urticales”. Both glandular and eglandular trichomes occur in the family. Among all the species examined, only P. grandis is completely glabrous and M. stipitata has a hairless perianth. The most common type of protective hair is simple, unicellular and eglandular, which occurs on the perianth margin or surface of many species like S. soleirolii, G. arborea, P. judaica and R. humilis, as well as the receptacle of F. tenacissima. The tepals of U. dioica are covered by modified unicellular eglandular conical trichomes with rough surface. This type of hair is also present on the gynoecium surface of M. stipitata and P. zeylanica. Notably, it also appears on the stigma of P. judaica and G. arborea as well as P. zeylanica, either surrounding or alongside the stigmatic trichomes. Unicellular, hooked trichomes appear on the bract/bracteole surface of S. soleirolii. L. aestuans is special in bearing only multicellular capitate glandular trichomes on the out surface and margins of the tepals. Similar glandular trichomes are also present on the surface of S. soleirolii bracts and perianth. A mass of threat-like hairs covers the surface of gynoecium in F. tenacissima and the surface of perianth in Cecropia sp. In the male (bisexual) flowers of S. soleirolii, G. arborea and P. judaica, numerous multicellular, filiform hairs arise from the receptacle surrounding the pistillode (gynoecium) and the stamens.

4.4 Evolution of sexuality in Urticaceae The basic form of the flower in Urticaceae is unisexual and monoecious, whereas bisexual flowers occur in some groups of the tribe Boehmerieae, such as Parietaria and Pouzoulia. There are generally two hypotheses on the evolution of the bisexual flower in Urticaceae: 1. Unisexual flowers are the ancestral character state, and bisexual flowers are formed by compaction and reduction of unisexual inflorescences on monoecious individuals; 2. Bisexual flowers are the ancestral character state and unisexual flowers are generated by reduction and partial abortion of bisexual flower (Mitchell & Diggle 2005).

For the first hypothesis, the compact and bisexual inflorescence pattern is a favorable indicator. Inflorescences with female flowers on top and male flowers at the bottom, or sprays with axillary female inflorescences and male inflorescences around can be a primitive stage that has the possibility to form a bisexual flower through shortening the axes, reducing the axes number and flower number, and abortion of the female perianth. An ideal model for this within the family would be Forsskaolea or Droguetia in the tribe Boehmerieae, both of which 48 possess an inflorescence with apetalous female flowers in the middle surrounded by uni- stamened male flowers (Friis 1992) (Fig. 7G). And the female flowers of F. tenacissima even bear a long and conspicuous gynophore (Fig. 7H). Also the molecular work has showed that the most common bisexual genus Parietaria is not genetically far from the Forsskaolea branch (Wu et al. 2013). Thus Forsskaolea and Droguetia might be an intermediate stage during the evolution in this theory. Considering the high frequency of the present of a gynophore (Fig. 6G, 7B, 7H, 20A; Table in Appendix) and the unique occurrence of bisexual flower within this tribe, it is possibly that the gynophore is originated from a pedicel remnant of the ancestral female flower. However, this cannot explain the present of a gynophore in the female flowers of Parietaria, Gesnounia, Soleirolia and even Cecropia, because they normally would not possess a pedicel remnant within the perianth whorl. Nor can it explain the consistency of the gynoecium with a single carpel and only one basal ovule in all Urticaceae or the universality of a pistillode in the male flowers.

The second hypothesis is more straightforward and is prevalent in angiosperm evolution, generating a new type of flower with organ residues (Mitchell & Diggle 2005). This theory corresponds with the phylogenetic fact that Urticaceae is the most advanced group within the urticalean lineage while Ulmaceae is the most basal group (Savolainen et al. 2000a; Savolainen et al. 2000b; Soltis et al. 2011). Because bisexual flowers are frequent in Ulmaceae whereas unisexual flowers by abortion are indistinctly differentiated between males and females, and in all the other groups of the “Urticales” the predominant character state is unisexual (Berg 1977). Besides, the universality of the pistillode in male flowers and the staminodes present in P. grandis can be evidence for supporting this theory. Friis (1992) believed that the bisexual flower in Parietaria is a preservation from the ancestral stage. Evidence can be that the basic number of chromosomes in Parietaria is x=7 or x=8, which seems more primitive, compared to the other groups where the basic chromosome number varies from x=10 to x=20 (Le Coq 1963; Fedorov 1969 in Friis 1992). However, it is still a bit problematic when explaining the relationships within Parietaria-Gesnouinia-Soleirolia clade. In the molecular phylogeny, Soleiroli is the basal-most genus among the three, while Paritaria and Gesnouinia are more derived (Wu et al. 2013). Morphologically, all the three genera have structurally bisexual flowers but only functionally bisexual in Parietaria. In the latter two, the gynoecium goes through an almost integrated developmental process and is 49 well differentiated into an ovary and a style, except for an abnormal stigma without functional trichomes. The stigma is even more reduced in Soleirolia than in Gesnouinia (Fig.6E, 7F).

Considering all above, the fact that the unisexual flowers of Urticaceae are evolved from bisexual ancestors with a residue of the pistil is the more likely hypothesis. However, there are occasional shifts back to bisexual forms simply by a redevelopment of the pistillode with a functional stigma. In this case, the morphological variations among different lineages, particularly the Parietaria clade in this study, can be quite nicely explained with the molecular phylogenies.

4.5 Pollination syndromes of Urticaceae Members of Urticeae, Elatostemeae, and Boehmerieae (i.e. Urticaceae s.s.) are strictly wind-pollinated (Berg 1977; Berg 1989). The adaptations to anemophily in these clades are well defined. The flowers are tiny and almost greenish, and the inflorescences are always densely clustered panicles or catkins, as most of the other anemophilous groups. In male flowers, the filaments are basically slender and the anthers are often relatively large (Berg 1989). The most elaborated adaptation is the inflexed stamens bending outwards suddenly at anthesis and sheding the pollens. This character also occurs in some genera of Moraceae and Ulmaceae, but not as universal as in Urticaceae (Berg 1977). In female flowers, the trichomatic stigma is the most effective adaptation to pollen capture. Besides, the special tentacular hairs derived from the integuments are very likely to have a special function in the pollinating process. It could be functional in identifying pollens from the correct species because one of the major problems for wind-pollinated species is the difficulty in restricting the pollen resources. Although the pollen recognition usually takes place at the stigma, however, it is still feasible to suppose a final restriction inside the ovary (Heslop-Harrison & Shivanna 1977; Heslop-Harrison & Heslop-Harrison 1985; Elleman et al. 1992; Sharma & Bhatla 2013).

Cecropieae is different from the other three clades in being predominantly insect- pollination and doesn’t have the characteristic inflexed stamen as all the other Urticaceae do, except Cecropia. Cecropia is a special case in Cecropieae because it much more resembles the Urticaceae s.s. in the pollination mechanism (Berg 1977; Berg 1978). It is the only wind- pollinated genus within the clade. In many species of Cecropia, “the anther is detached from 50 the straight filament by abscission, but it remains loosely attached to the flower (by the sticky ends of the appendices of the anther or by a bundle of stretched spiral thickenings of the tracheary elements of the stamina vascular bundle) and is movable” (Berg 1977). The slender pendulous spike and a periodic reproductive phenology coordinated with climate seasonality (Zalamea et al. 2011) are also typical adaptations to anemophily.

4.6 Indications of infra-familial and interfamily relationships of Urticaceae Infra-familial relationships In the representative species studied here, all kinds of female variations occur among different tribes and it is still hard to summarize any tribal characters with such a limited number of species involved. However, based on morphological observations in this study and other literature references, regular patterns of female variations can still be summarized to some degree. In general, Urticeae and Elatostemeae tend to have free tepals, while Boehmerieae and Cecropieae have a large portion of species possessing a tubular perianth (Wu et al. 2015; Table 2). Elatostemeae is the least hairy tribe, especially Pilea, where nearly all the species are hairless, and rather variable in perianth merism. Cecropieae is characterized by strict dioecy and the absence of inflexed stamens in relation to insect-pollination. Yet the detached anthers and the pendulous inflorescence in Cecropia are considered as wind- pollinated syndromes. Boehmerieae has the largest diversity in floral characters. Parietaria, Gesnouinia, and Soleirolia examined here forms a well-supported branch both in the morphological and molecular study. Parietaria and Gesnouinia are more similar in the form of inflorescence while Soleirolia has solitary flowers. All the three genera possess a much more developed gynoecium in male flowers, compared to other groups in Boehmerieae, although only in Parietaria the gynoecium is functional while in the other two it is pistillode.

Interfamily relationships Apetaly is a common character in Rosales, especially in the urticalean branch, but in Rosaceae and Rhamnaceae, biseriate perianth occurs more frequently. The petals present within the order are believed to be a regained character originated from the outmost stamens by homeiosis. And under this theory, apetaly is the ancestor state for the order. I have discussed earlier that the nature of the perianth in Urticaceae is most likely to originate from a calyx, and no trace of corolla can be found in all the species studied here, which in a way 51 supports the theory of the secondarily derived acquisition of a corolla in Rosaceae and Rhamnaceae.

The ancestral character of the gynoecium in the urticalean lineage is a bilocular ovary derived from two (or three) carpels with several ovules, and one of the carpels is always suppressed (Bechtel 1921; Berg 1989). The anatomical study of the flower vasculature in Urtica indicated that it has a similar vascular supply of pistil to that of some genera in the sister families (e.g. Ulmus, Morus), which is related to the psedomonomerous ovary formation from two carpels (Bechtel 1921). However, compared to the other sister families, Urticaceae is much more like “unicarpellate” because all the species have only one stigma/style and an orthotropous basal ovule (Weddell 1854; Friis 1989). On the other hand, Moraceae, Ulmaceae and Cannabaceae all have an anatropous (sub)apical ovule and even bear a second stigma/style from the abortive carpel in some groups, such as Ficus, Morus, Ulmas and Cannabis (Bechtel 1921; Corner 1962; Cramer & Swann 1966; Berg 1989). Berg (1989) interpreted the uniqueness of Urticaceae as a more derived character from the latter. This hypothesis is accordant with the molecular phylogeny of the former “Urticales” and viable in the floral development. When comparing the early gynoecium development between Urticaceae and cases of Cannabaceae and Moraceae in Cramer and Swann’s work (1966), they seem to share a very similar initiation pattern of the ovule and ovule wall. The differences lie in the later process when the ovary wall continues to grow unequally. In the case of Ficus, Morus and Cannabis, the extension growth happens on both sides of the primordium forming two carpels with only one can be complete developed and thus two grooves or scars by fusion should be seen theoretically on the ovary wall in these groups. In Urticaceae, however, the suppressed side of ovary wall is completely aborted, and no sign of the second carpel is formed during the whole developmental process.

5 Conclusion

The morphological variation of Urticaceae flower is rather diverse, especially in female flowers. Male flowers Urticaceae s.s. are less variable and generally tetramerous with inflexed stamens and a pistillode. In Cecropia, the stamens are straight but the anthers are detached from the filaments. In female flowers, the perianth shows different merism in different groups and appears as either free tepals or more or less fused tubes or is even absent. The perianth development process of examined flowers demonstrates that the tepals are from sepal origin and supports the view of apetaly being the ancestral state of the lineage. The stigma formed by a cluster of multicellular or unicellular trichomes has a variety of shapes and tends to become elongated and penicillate in Boehmerieae and Cecropieae. The pseudomonomerous carpel of Urticaceae differs from that of Moraceae or Cannabaceae in the complete abortion of the second carpel at the beginning of gynoecium development, which is a more derived character compared to the sister groups in urticalean branch. A grooved scar is frequently present as a result of the carpel folding from one side to the other. The ovule of Urticaceae species is basal and orthotropous with two integuments that have a tendency to derive trichomatic apex around the mycropyle, especially in Myriocarpa. The function of the hairs at the micropyle is not clear but very likely to be associated with pollen tube inducing and identification. Bisexual flowers occur only in Boehmerieae, where a gynophore is frequently present and can be interpreted as an indication to a reduced inflorescence relating to bisexual flower formation. The basic inflorescence types are unisexual cymose panicles, but can derive into multiple variations in relation to different sexuality, reduction or fusion of axis and modifications of the inflorescence receptacle. The inflorescence form may also have influence on the evolution of bisexual flowers within the family. The trichomes appeared in the family are different shape, structure and positions, and can be classified into stigmatic trichomes and protective hairs.

The morphological character changes do not always correspond to the molecular phylogeny. However, there are still character tendencies observed from those samples that correspond to the molecular data, such as the perianth merism, stigma shape, presence of gynophore and trichome distribution. In particular, the Soleirolia-Gesnouinia-Parietaria lineage shows a high accordance between the molecular phylogeny and morphological variations, especially in the comparison of the inflorescence type and the pistillode in male 53 flowers. A secondarily derived acquisition of bisexual flowers from the redevelopment of the pistillode of those functionally male flowers is a feasible theory for this clade.

The systematic position of Urticaceae as an advanced group sister to Moraceae is supported by the floral development revealed in this study. The gynoecium formation of the Urticaceae species resembles that of Moraceae and Cannabaceae in Cramer and Swann’s study (1966) of flower ontogenesis, but the absence of a residue of the secondary carpel in Urticaceae indicates that this character is more derived and that Urticaceae is fairly advanced within the lineage.

Useful further work for the family could be more detailed floral developmental observations involving more species covering a larger span of developmental stages because some of the species studied here still lacks important stages during the whole development and the mature form of stigma and ovule need to be carefully examined in older flowers. Anatomy and chemical tests may be needed to reveal any possible connections between the stigmatic trichomes and the ovule hair appendages. The receptibility or fertility of the well- developed pistillode in Gesnouinia and Soleirolia needs to be tested to confirm whether they are functionally male for sure or possibly bisexual in some cases and to give further clues to the formation of bisexuality in the family.

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Appendix

Table 2. Summary of the major characters in mature female flowers Perianth Gynoecium Special Name Merism Tepal fusion Symmetry Stigma shape Stigmatic trichomes Ovary groove Gynophore Urtica dioica 4 Free Disymmetry Capitate- Multicellular Absent Absent filiform Laportea aborea 4 Free Monosymmetry Capitate Multicellular Absent Absent

Myriocarpa stipitata 2 Basal fused Disymmetry Spatulate Multicellular Absent Absent

Pilea grandis 3 Free Monosymmetry Capitate Unicellular Present Absent

Soleirolia soleirolii 3-4 Tubular Polysymmetry Capitate Unicellular Present in male Present

Gesnouinia arborea 4 Half fused Polysymmetry Penicillate Unicellular Present Present

Parietaria judaica 4 Basal fused Polysymmetry Capitate Unicellular Present Present

Forsskaolea tenacissima Abscent Abscent Abscent Capitate Unicellular ? Present

Rousselia humilis 2 (lobed) Tubular Disymmetry Capitate Unicellular ? Absent

Pouzolzia zeylanica 2 (lobed) Tubular ? Penicillate Unicellular ? Absent

Cecropia sp. ? Tubular Disymmetry Penicillate? Multicellular *Present Present

?: Not observed in this study; * Material not old enough.