Anther and Gynoecium Structure and Development of Male and Female Gametophytes of Koelreuteria Elegans Subsp

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Flora 255 (2019) 98–109

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Anther and gynoecium structure and development of male and female gametophytes of Koelreuteria elegans subsp. formosana (Sapindaceae): T Phylogenetic implications ⁎ Adan Alberto Avalosa,1, Lucía Melisa Zinia,1, María Silvia Ferruccia, Elsa Clorinda Lattara,b, a IBONE-UNNE-CONICET, Sargento Cabral 2131, C.P. 3400 Corrientes, Argentina b Cátedra de Morfología de Plantas Vasculares, Facultad de Ciencias Agrarias, Sargento Cabral 2131, C.P. 3400 Corrientes, Argentina

ARTICLE INFO ABSTRACT

Edited by Louis Ronse De Craene Anther and gynoecium structure and embryological information in Koelreuteria and Sapindaceae as a whole fl Keywords: remain understudied, as well as the evolution of imperfect owers in the latter. The aims of this study were to Monoecy analys in K. elegans subsp. formosana the anther and gynoecium structure and the development of male and Microsporogenesis female gametophytes in the two floral morphs of putatively imperfect flowers. Standard techniques were applied Orbicules for LM and SEM. Compared to the normal anther development in staminate flowers, a delayed programmed cell The pollen tube transmitting tract death of tapetum, septum and middle layers on the onset of microspore stage result in indehiscent anthers in the Ovule campylotropous functionally pistillate flowers. Orbicules are reported for the first time in Sapindaceae. Gynoecium development Character evolution in functionally pistillate flowers is normal, whereas in functionally staminate ones a pistillode with degenerated ovules at the tetrad stage is formed. The pollen tube transmitting tract consists of one layer of epithelial cells with a small lumen in the style and ovary. The anatomy of the latter revealed an axile placentation and complete septum. Reproductive characters of Koelreuteria shared with other Sapindaceae taxa are a secretory binucleate tapetum, simultaneous cytokinesis, unicellular stigmatic papillae, Polygonum type megagametophyte with ephemeral antipodals, and ovules campylotropous, with funicular obturator and amphistomic micropyle. Imperfect flowers in the family are triggered by defective sporophytic tissues impacting on the normal development of microspores and megaspores, disrupting embryological events in staminate and pistillate organs at different developmental stages. Reconstructing ancestral character state under parsimony revealed that two ovules per locule, absence of obturator, presence of hypostase, and binucleate tapetum may be regarded as plesiomorphic for Sapindaceae. These analyses have provided information on the evolution of embryological characters in the family and its phylogenetic significance.

1. Introduction Based on molecular phylogenetic studies, Sapindaceae s.l. comprises about 1900 species and 141 genera distributed among four subfamilies: The systematics of the family Sapindaceae has been approached Dodonaeoideae Burnett, Hippocastanoideae Burnett, Sapindoideae from different fields of research including macromorphological and Burnett and Xanthoceratoideae Thorne & Reveal. (Harrington et al., palynological characters (Radlkofer, 1931-1934; Müller and Leenhouts, 2005; Thorne and Reveal, 2007; Buerki et al., 2009, 2011; Acevedo- 1976), floral organogenesis (Cao et al., 2018), embryology (Cao et al., Rodríguez et al., 2011; Cole and Ferrucci, 2018). Nevertheless, the 2008; Zhou and Liu, 2012; González et al., 2017), paleontology (Wang phylogenetic relations for many clades still remain unclear and thus the et al., 2013) and molecular analyses (Harrington et al., 2005; Buerki reproductive anatomy and embryological data would be a useful fra- et al., 2009, 2010, 2011), despite these lines of evidence the systematics mework to help clarify them. of Sapindaceae are still subject of discussion. The overall advances in Sapindaceae has mostly monoecious species, dioecious or less fre- the molecular phylogenetics of the family were not accompanied by a quently polygamous species (Acevedo-Rodríguez et al., 2011). Floral robust basis of morphological knowledge to identify distinctive features dimorphism is a widespread trait in this family. Thus, the presence of that may be putative valuable synapomorphies (Buerki et al., 2010). staminate flowers with rudimentary gynoecium and perfect flowers that

⁎ Corresponding author at: IBONE-UNNE-CONICET, Sargento Cabral 2131, C.P. 3400 Corrientes, Argentina. E-mail address: [email protected] (E.C. Lattar). 1 These authors contributed equally to the manuscript. https://doi.org/10.1016/j.ﬂora.2019.04.003 Received 26 October 2018; Received in revised form 16 March 2019; Accepted 12 April 2019 Available online 18 April 2019 0367-2530/ © 2019 Published by Elsevier GmbH. A.A. Avalos, et al. Flora 255 (2019) 98–109

Fig. 1. Androecium in Koelreuteria elegans subsp. formosana. A. Comparison between stamens of functionally pistillate (left) and staminate (right) flowers. B. Stamen of pistillate flower. C. Stamen of staminate flower. D. Detail of anther epidermis. E. Longitudinally dehiscent anthers of staminate flower. F. Inner surface of the anther locule with Ubisch bodies (arrows). Scale bars: 1 mm (A), 200 μm (B), 500 μm (C), 20 μm (D), 100 μm (E), 5 μm (F).

are functionally pistillate (because they have nonfunctional stamens) is through an ethanol series with a pre-impregnant rinsing of tertiary common. Despite the fact that Sapindaceae is diverse in terms of species butyl alcohol (Gonzalez and Cristóbal, 1997) and infiltration in Histo- number, the studies on the floral morpho-anatomy and/or embryology plast® paraffin (Biopack, Buenos Aires, Argentina), according to were limited to only a few species (Nair and Joseph, 1960; Khuslani, Johansen (1940). Flowers were sectioned transversely and long- 1963; Karkare-Khushalani and Mulay, 1964; Gulati and Mathur, 1977; itudinally (10–12 μm thick) with a Microm HM 350 rotary microtome Mathur and Gulati, 1980, 1981, 1989; Ha et al., 1988; Ronse Decraene (Microm International, Walldorf, Germany). The sections were stained et al., 2000; Weckerle and Rutishauser, 2005; Cao et al., 2008; Solís with astra blue–safranin (Luque et al., 1996) and permanently mounted et al., 2010; Zhou and Liu, 2012; Zini et al., 2012; González et al., 2014, with synthetic Canada balsam (Biopur, Buenos Aires, Argentina). 2017; Avalos et al., 2017; Cao et al., 2018), and most of the information Temporary slides of fresh mature pollen grains were created to test derived from these studies have not been included in morphological pollen viability using acetocarmine (Radford et al., 1974). Morpholo- characterizations of groupings supported by the molecular phylogeny. gical and anatomical features were observed and photographed using a Koelreuteria Laxm. is a genus of monoecious trees native to eastern Leica MZ6 stereomicroscope and a Leica DM LB2 compound microscope Asia (Radlkofer, 1931–1934) and cultivated as ornamentals in nu- (Leica, Wetzlar, Germany), both equipped with a digital camera (Leica merous gardens of the world (Meyer, 1976; Cao et al., 2018). The digital). The morphology of the androecium and gynoecium was stu- systematic position of the genus in Sapindaceae has been changing over died with scanning electron microscopy. For SEM, fixed flowers were time according to the criteria used by different authors (Cao et al., dissected and dehydrated through a series of increasing acetone solu- 2018). Molecular phylogenetic analysis proposed by Buerki et al. tions. The material was then critical point-dried with solvent-sub- (2009) reveals that Koelreuteria integrates an early-diverging lineage of stituted liquid carbon dioxide and sputter coated with gold-palladium. the subfamily Sapindoideae which is characterized by both ancestral Orbicules were observed from mature anthers; and were also dehy- and derived characters. Conversely, Cao et al. (2018) found evidence in drated through a series of increasing ethanol solutions, air drying, and floral organogenesis that reflects affinities between Koelreuteria and the then coated with gold-palladium. Photomicrographs were obtained subfamily Dodonaeoideae. Hence, it is evident that knowledge about using a JEOL 5800 L Vat 20 kV (JEOL USA, Peabody, MA, USA). the floral traits in this genus would be useful to understand its sys- To visualize the possible evolution of selected embryological char- tematic position within Sapindaceae. acters, a tree topology of the Sapindaceae phylogeny modified from The present study aimed to analys the anatomy of the anther and Cole and Ferrucci (2018) was used. This topology is based on a con- gynoecium in K. elegans subsp. formosana (Hayata) F.G. Mey. and sensus of parsimony, maximum likelihood and Bayesian analyses. The characteris stages of embryological development focusing on i) the tree topology was assembled by hand using Mesquite v. 3.40 (Maddison comparative analysis between both pistillate and staminate flowers and and Maddison, 2018), and it was rooted to a designated outgroup which the floral sexual differentiation in imperfect flowers, and ii) the phy- is sister to Sapindaceae, a clade comprising Anacardiaceae, Burser- logenetic implications of embryological features for Sapindaceae. aceae, and Kirkiaceae (Muellner et al., 2007). Maximum parsimony was used to reconstructs the ancestral state of characters included in the data matrix. All characters were treated as unordered. The character 2. Materials and methods state codes and data matrix are provided in Appendix A.

Floral buds and flowers of both floral morphs were collected at different developmental stages on trees cultivated of K. elegans subsp. 3. Results formosana in Corrientes city, Argentina (27°'S, 58°'W), and fixed in FAA (formalin, acetic acid, ethanol). We analyzed three cultivated in- 3.1. Androecium morphology dividuals. The voucher specimens were deposited in the herbarium of the Instituto de Botánica del Nordeste (CTES), Argentina. For preparing In both floral morphs, the Bauplan is essentially similar and the histological sections, the material was processed by dehydration flowers have eight free stamens, the filaments are puberulus to

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Fig. 2. Microsporangium at different developmental stages in Koelreuteria elegans subsp. formosana.A–G Features shared by both floral morphs. A. Cross-section of the young anther at early stage of microspore mother cells, note the callose. B. Tetrasporangiate anther. C. Detail of the anther wall layers and microspore mother cells surrounded by callose. D. Late stage of microspore mother cells with a detail of binucleate tapetum. E. Anther wall layers and tetrahedral tetrads of microspores (arrow). F. Early free microspores (arrows). G. Late free microspore stage. H–K Features of male flowers: H. Anther at young pollen grain stage. I. Detail of anther showing endothecium with secondary wall thickenings (arrows) and remnants of tapetal cells Anther at mature pollen grain stage. Note the parenchymal septum broken (arrow). K. Dehiscent anther with a detail of pollen grains stained with acetocarmine.O: Features of functionally female flowers. L. Anther at young pollen grain stage with complete septa. M. Anther wall at young pollen grain stage, showing papillate epidermis, endothecium with wall thickenings (arrows), middle layers and complete tapetum layer. N. Anther at mature pollen grain stage, showing densely stained remnants of tapetum, shriveled but unbroken septum, nonfunctional stomium (asterisk), and pollen grains (arrows), detail of pollen nonfunctional grains stained with acetocarmine. O. Indehiscent anther at post-anthesis stage, note the pollen grains ag- glomerated (arrow). Abbreviations: e, epidermis; en, endothecium; ml, middle layers; mmc, microspore mother cells; s, septum; t, tapetum. Scale bars: 50 μm (A, B, I M, N, O), 25 μm(C–G), 100 μm (H, K, L).

pubescent, anthers are basifix, bithecal, oblong, puberulus (Fig. 1A–C, radially and the middle layers are crushed (Fig. 2E). The tapetum is of E) with papillose epidermis and striated cuticle (Fig. 1A–C, E, D). secretory type (Fig. 2E). Callose degradation release young free mi- Functionally staminate flowers have long exerted stamens and anthers crospores, which have a densely stained cytoplasm (Fig. 2F). During with longitudinal dehiscence (Fig. 1A, C, E), whereas the functionally microspore vacuolisation, the endothecium cells continue to increase in pistillate flowers have smaller stamens, and indehiscent anthers length and the secondary wall thickenings begin to develop. The middle (Fig. 1A, B). layers are still observed but are degenerated (Fig. 2G). The epidermis is slightly compressed and the fibrillar thickenings of the endothecium are 3.2. Anther structure, microsporogenesis, microgametogenesis and orbicules fully developed. At the young pollen grain stage, the two par- enchymatous septa separating pollen sacs in each theca break down In functionally staminate flowers the young microspore mother cells (Fig. 2H–J). At the mature pollen grain stage the stomium cells disin- (mmcs) differentiate from the sporogenous tissue, and become poly- tegrate and the anther dehisces through longitudinal slits (Figs. 1E, 2 gonal and surrounded by callose (Fig. 2A). During maturation, micro- K). In the functionally staminate flowers, tricolporate pollen grains are spore mother cells become rounded and separate from one another shed at two-celled stage, and they stained dark red with acetocarmine, (Fig. 2B–C). At this stage, the anther wall consists of an epidermis, an indicating that are viable (Fig. 2K). endothecium with thin-walled cells and conspicuous nuclei, two middle Under scanning electron microscope, the inner surface of the de- layers and a binucleate tapetum (Fig. 2C, D). Meiosis of the microspore hisced anther locules exhibits orbicules on the tapetal membrane. These mother cells is simultaneous and the resultant tetrads are of tetrahedral corpuscles are approximately in diameter, spheroidal or subspheroidal, configuration. Each microspore in the early tetrad is uniformly sur- and exhibit a smooth surface (Fig. 1F). The orbicules can be solitary or rounded by callose (Fig. 2E). The endothecium cells begin to enlarge they can merge, forming small aggregations (Fig. 1F).

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Fig. 3. Gynoecium morphology at anthesis in functionally pistillate ﬂowers of Koelreuteria elegans subsp. formosana. A. General view of the style and stigma (arrows). B. Detail of the stigma, showing two receptive areas (arrows) and a non-receptive zone (nrz). C. Style and stigma twisted (arrow) in a post-anthesis stage. D. Position of the young seeds in each locule. Scale bars: 2 mm (A, D), 1 mm (B), 4 mm (C).

In functionally pistillate flowers, the epidermis and endothecium stigmatic and stylar regions since they are in contiguity with the three are similar to those described in functionally staminate flowers, but the locular cavities, forming a small lumen at the center of the style middle layers, tapetum, and septa do not show notable signs of de- (Fig. 5B, C). This tissue is one cell layer thick, with tightly appressed gradation (Fig. 2L–M). Tapetal cells do notably decrease in volume and epidermal secretory cells ( Fig. 5B, C, I, K, L). Thus, the style is open, in the two septa in thickness than in the previous stages, but they do not transverse sections the stylar pttt is folded, tri-radiate and alternates break down. The stomia also remain intact, and therefore, anthers are with the locular cavities (Fig. 5C). This compitum culminates at the indehiscent (Fig. 2N). Pollen grains are often observed clustered within transition zone between symplicate into synascidiate parts (Fig. 5D). the locules, forming a compact mass. Some pollen grains exhibit a The pttt splits into three separate tracts in the center of the ovary, one normal appearance, whereas others show abnormal shapes or are per locule (Fig. 5J). The inner epidermis of the ovary is covered with completely collapsed, the cytoplasm of the pollen grains stained weakly multiseriate glandular trichomes comprising a two-celled stalk and a with acetocarmine, indicating that they are not viable (Fig. 2N–O). The multicellular head (Fig. 5G). Placentation is axile, and each placenta androecium in functionally pistillate flowers persists at the early stages bears two syntropous superposed ovules (i.e. curved away from the rim of fruit formation. of the carpel; Endress, 1994), (Fig. 3D). Gynoecium of functionally staminate flowers is a three-carpellate pistillode with a differentiated stigma, a short style, and an ovary 3.3. Gynoecium structure at anthesis containing ovules (Fig. 6A–C). The stigmatic region is covered by unicellular papillae (Fig. 6D), and the style and ovary are anatomically fl In functionally pistillate owers, the gynoecium is syncarpous and similar than those of functionally pistillate flowers (Fig. 6C–D). three-carpellate. The stigmatic region consists of three lateral receptive surfaces, covered with unicelullar papillae (Figs. 3A, B, 4 A). Along the stigmatic region, these three lateral zones alternate with three non re- 3.4. Ovule and female gametophyte ceptive area, which consist of flat epidermal cells with striated cuticle (Figs. 3B, 4 B, 5 A). The stigma is of similar length as the style (Figs. 3A, The ovule in functionally pistillate flowers is bitegmic and campy- C, 4 A). After anthesis, both the stigma and the style are twisted and lotropous, and is vascularized by a single vascular bundle that ends in change color, from yellow to brownish in the former and from reddish the chalaza (Fig. 7A). The outer and inner integuments are six to seven to yellow in the latter (Fig. 3A, C). and four to five cell layers thick, respectively. There is a zig-zag mi- The stigmatic region is asymplicate. Carpels are united postgenitally cropyle formed by both integuments (Fig. 7B). The funicular obturator by interlocking ventral epidermal cells (Fig. 5A). The hemisymplicate is approximately one-quarter as long as the ovule (Fig. 7A). It has a region below encompasses the end of the stylar region down to the secretory epidermis consisting of short papillose cells with densely upper portion of the ovary (Fig. 5B, C) and, which is otherwise almost stained cytoplasm and conspicuous nucleus (Fig. 7B). Epistase and entirely synascidiate, which extends down to the base of the ovary hypostase are absent. – (Fig. 5D G). Externally, the abaxial median region of each carpel pre- The ovule is crassinucellate and the megaspore mother cell is deeply sents a deep longitudinal furrow along the style and ovary (Fig. 4A) and buried under several cell layers. Embryo sac development corresponds has a single dorsal vascular bundle (Fig. 5C). The stigmatic papillae to the Polygonum type. The egg cell is distinguished by the presence of connect with the ventral side of the fused carpels (Fig. 5A, B, H). a micropylar vacuole and a chalazal nucleus (Fig. 7C). The synergids The style is glabrous, long; the epidermis is composed of rectangular present a slightly developed filiform apparatus (Fig. 7D). The central cells with striated cuticle (Fig. 4C). The style has one-layered epidermis, cell contains a secondary nucleus and a vacuole (Fig. 7E). Antipodals celled-layers of parenchyma consisting of polygonal cells occasionally are ephemeral (Fig. 7F). with druses. The ovary is hairy (Fig. 4D). At the base of the stigma, In ovules of functionally staminate flowers, the nucellus and in- there are three pollen tube transmitting tracts (pttt) situated between teguments become atrophied before they complete their curvature and the pairs of ventral vascular bundles of adjacent carpels (Fig. 5C, I, K). degenerate at the linear tetrad stage (Fig. 6E, F). The pttt forms an internal compitum throughout the entire length of the

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Fig. 4. Gynoecium morphology in functionally pistillate ﬂowers of Koelreuteria elegans subsp. formosana. A. View of the entire gynoecium; note the median dorsal wall furrow of a carpel (arrow). B. Level B in A; detail of the stigma with two receptive lateral areas (arrows) covered with unicellular papillae and two non-receptive dorsal zones (asterisks). C. Level C in A; detail of the style epidermis. D. Level D in A; detail of the outer ovary wall covered with unicellular hairs. Scale bars: 500 μm (A), 20 μm (B, C, D).

3.5. Character evolution tissue, plays a nutritional role and supplies the pollen grains in the formation of various enzymes and precursors of the exine (Pacini et al., The most parsimonious reconstructions for Sapindaceae are, ab- 1985; Chapman, 1987; Pacini, 1990; Pacini and Franchi, 1993). In K. sence of obturator, presence of hypostase, binucleate tapetum, and elegans subsp. formosana, the tapetum is secretory. This tapetum type is tricolporate pollen (Fig. 8A-D, Appendix B). The ancestral state of the unique in the family and possibly in Sapindales, whereas the number of number of ovules per locule is equivocal for the family. In the clade nuclei in tapetal cells is a very variable character (Gulati and Mathur, comprising the subfamilies Hippocastanoideae, Dodonaeoideae and 1977; Mathur and Gulati, 1980, 1981, 1989; Ha et al., 1988; Cao et al., Sapindoideae the two possible states are two ovules or a single one. 2008; Solís et al., 2010; Zhou and Liu, 2012; Zini et al., 2012; González Within Sapindoideae, the state one ovule is reconstructed as ancestral et al., 2014, 2017). The tapetal cells in K. elegans subsp. formosana are for the sister group of Koelreuteria.(Fig. 8A). The absence of a hypostase binucleate and although variations exist among the species, the bi- would be apomorphic for the genus and for other three different nucleate condition was reported in most genera studied to date within lineages of the family (Fig. 8B). The obturator may be synapomorphic all the subfamilies of Sapindaceae (Table 1). for the clade consisting of the subfamilies Dodonaeoideae and Sa- Orbicules or Ubisch bodies are acellular structures of sporopollenin pindoideae, but it is reconstructed as equivocal in the node that in- that may occur on the inner tangential and radial walls of tapetal cells cludes Hippocastanoideae (Fig. 8C). The ancestral state for the micro- (Verstraete et al., 2014) or can be defined as corpuscles of various sizes pyle type (endostomic/amphistomic) is not resolved for Sapindaceae, that react similarly to pollen exine in staining, autofluorescence, and and is equivocal for most clades. Similarly, the ancestral ovule curva- resistance to acetolysis (Galati, 2003; Galati et al., 2010). The pro- ture is mostly inferred either as anatropous or campylotropous (Ap- duction of orbicules is one of the main features of the secretory tapetum pendix B). ( Pacini, 1990; Pacini and Franchi, 1993; Huysmans et al., 1998; Lattar et al., 2012, 2014). Orbicules in functionally male flowers of K. elegans subsp. formosana are spherical to subspherical and with a smooth sur- 4. Discussion face. According to Galati et al. (2010), this morphology is predicted for melitophylous species and coincides with field observations revealing 4.1. Anther structure: tapetum and orbicules visits of flowers by bees (Avalos unpublished data). In this study, the presence of orbicules in Sapindaceae is reported The tapetum, a specialized cell layer surrounding the sporogenous

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Fig. 5. Gynoecium structure at anthesis in functionally pistillate ﬂowers of Koelreuteria elegans subsp. formosana. A–G: Transverse sections. A. Stigmatic head with three receptive lateral areas (arrows) and three non-receptive dorsal zones. B. Stigmatic region basis showing three conﬂuent locular cavities (arrows) alter- nating with the pollen tube transmitting tract (asterisks) and three slits (arrowheads). C. Base of the style, showing the locules (arrows), pollen tube transmitting tract (asterisks), syn- laterals vascular bundles and the three external furrows. D. Upper part of the ovary with three locular spaces (arrows) and tri-radiate pollen tube transmitting tract (asterisks). E, F. Ovary above the placenta level, showing locules (arrow), septa and ovules. G. Ovary at the placenta level, note multicellular glandular trichomes in the inner epidermis (arrows) and a detail of the trichome. H–L: Longitudinal sections. H. Stigmatic head with a detail of the unicellular papillae. I. detail of the solid style, note the locule cavities (arrows) and the pollen tube transmitting tract Upper part of the ovary, showing pollen tube transmitting tract and syntropous ovule. K. Detail of pollen tube transmitting tract below the stigma, note the dorsal vascular bundle. L. Detail of stylar pollen tube transmitting tract. Abbreviations: f, furrow; fu, funicle; nrz, non receptive zone; o, ovule; pttt, pollen tube transmitting tract; vb, dorsal vascular bundle. Scale bars: 100 μm (A–D, I), 200 μm(E–H, 50 μm (K, L).

for the ﬁrst time. Therefore, it would be interesting to extend this kind Craene et al., 2000; Weckerle and Rutishauser, 2003, 2005), but it is of studies to other members of the family in order to test the systematic unknown in most genera of the family. Other Sapindalean families value of orbicules and to verify their occurrence among the clades re- exhibiting the same stigma are Aceraceae (Peck and Lersten, 1991a,b), cognized in the current classiﬁcation. Xanthoceraceae (Zhou and Liu, 2012), several Rutaceae (de Souza et al., 2004; Heslop-Harrison and Shivanna, 1977; Ramp, 1988; Caris 4.2. Gynoecium structure at anthesis et al., 2006), and Nitrariaceae (Bachelier et al., 2011). The uniseriate multicellular stigmatic papillae evolved only in the clade of Anacar- The presence of a three-carpellate gynoecium as observed in K. diaceae, Burseraceae, and Kirkiaceae (Bachelier and Endress, 2008, elegans subsp. formosana is a widespread feature among the subfamilies 2009). As in K. elegans subsp. formosana, the stylar pttt consisting of an of Sapindaceae (Zhou and Liu, 2012). Furthermore, a stigmatic region epithelium that delimits a narrow central lumen with secretion has also formed by the postgenitally united free carpel tips is a feature con- been reported previously in species of Averrhoidium, some Cardios- sistent with the family as well as with other taxa of the Sapindales permum, Paullinia and Serjania. However, variations may occur at the (Burseraceae, Kirkiaceae, Bachelier and Endress, 2008, 2009; Nitrar- species level in Paullinia since other two types of pttts were described, iaceae, Ronse De Craene and Smets, 1991; Bachelier et al., 2011; Me- i.e. a wide canal delimited by hairs or a solid tract of several layers of liaceae, Gouvêa et al., 2008a, b; Rutaceae and Simaroubaceae, Endress elongated secretory cells (Weckerle and Rutishauser, 2003, 2005). In et al., 1983). A stigmatic surface with unicellular papillae was observed agreement with Ronse De Craene et al. (2000), this study shows that the in this work, and was previously reported in K. paniculata Laxm., spe- so-called septal cavities within the style of Koelreuteria may correspond cies of Averrhoidium Baill., Cardiospermum L., Paullinia L., Serjania Mill. to pttts (with a large folded inner surface lined with secretory tissue) and Urvillea Kunth (Heslop-Harrison and Shivanna, 1977; Ronse De which are continuous with the papillate stigma.

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Fig. 6. Gynoecium morpho-anatomy at anthesis in staminate ﬂowers of Koelreuteria elegans subsp. formosana. A. Pistillode in an anthetic ﬂower (arrow). B. Detail of stigma and style (arrow). C. Transversal section of the ovary, showing three aborted ovules (arrows). D. Longitudinal section of the stigma and the pollen tube transmitting tract formed by postgenital fusion (asterisk). E. Ovule with linear tetrad of megaspores (arrows). F. Ovule with atrophied integuments and three micropylar aborted megaspores (arrows). Abbreviations: ii, inner integument; oi, outer integument. Scale bars: 2 mm (A), 1 mm (B), 200 μm (C), 20 μm(D–F).

Fig. 7. Ovule and female gametophyte in- functionally pistillate flowers of Koelreuteria elegans subsp. formosana, paraffin sections stained with astra blue and safranin A. Longitudinal section of a mature ovule. B. Detail of funicular obturator and micropyle (arrows). C. Egg cell and the two synergids. D. Detail of a synergid with filiform apparatus. E. Secondary nucleus (arrow) of the central cell. F. Antipodals nucleus, with a detail of degenerated antipodals. Abbreviations: a, antipodals; ec, egg cell; fa, filiform apparatus; ii, inner integument; o, obturator; oi, outer integument; s, synergid; vb, vascular bundle. Scale bars: 50 μm (A, C, E, F, H, I), 20 μm (D), 100 μm (G), 200 μm (B).

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Fig. 8. Tree topology of Sapindaceae modiﬁed from Cole and Ferrucci (2018) showing maximum parsimony ancestral state reconstructions of four embryological characters. A. Ovule number. B. Hypostase. C. Obturator. D. Number of nuclei in tapetum cells.

Ovules of the Sapindoideae and Dodonaeoideae are campylo- Meyer (1976) and Ronse de Craene et al. (2000) reported it as parietal tropous, anatropous or hemianatropous, bitegmic, crassinucellate, and for other species of the genus. The latter authors state that the septa with obturator of funicular (as in K. elegans) or placental origin above the placentae are incomplete in K. paniculata. Therefore, the (Karkare-Khushalani and Mulay, 1964; Corner, 1976; Gulati and placentation in Koelreuteria seems to be a variable character. Mathur, 1977; Johri et al., 1992; Ye et al., 1992; Ronse De Craene et al., Finally, the female gametophyte of Polygonum type with presence 2000; Weckerle and Rutishauser, 2005; González et al., 2017). In our of ephemeral antipodals can be considered a predominant feature of the material, ovules are campylotropous, whereas in K. paniculata they are Sapindales (Nair and Joseph, 1960; Gulati and Mathur, 1977; Mathur anatropous (Ronse De Craene et al., 2000). The micropyle can be en- and Gulati, 1980, 1981, 1989; Ha et al., 1988; Johri et al., 1992; Ye dostomic (Dodonaea Mill., Delavaya Franch., Cardiospermum, Paullinia, et al., 1992; Cao et al., 2008; Zhou and Liu, 2012; González et al., 2014, Serjania, Urvillea, Allophylus L., and Magonia A. St. Hil., Karkare- 2017). However, the Penaea-type female gametophyte is restricted to Khushalani and Mulay, 1964; Weckerle and Rutishauser, 2005; Cao Biebersteiniaceae, which is sister to all other Sapindales (Yamamoto et al., 2014; González et al., 2014, 2017) or amphistomic (Filicium et al., 2014). Thwaites, Gulati and Mathur, 1977). However, an amphistomic micropyle of zigzag shape, as was observed here, has also been reported in fl ff Averrhoidium and within many other lineages of the Sapindales 4.3. Anatomical aspects of oral sexual di erentiation (Weckerle and Rutishauser, 2003; reviewed in Endress and Matthews, fl 2006; Bachelier and Endress, 2009; Bachelier et al., 2011). The ob- The mechanisms that could lead to imperfect owers in angios- ff turator prevails in Sapindaceae, and it also occurs in Acer and many perms are diverse and may occur at di erent stages, representing sev- Rutaceae (Weckerle and Rutishauser, 2005; Zhou et al., 2004; Zhou and eral distinct developmental transitions in the evolution from her- fl Liu, 2012; Ramp, 1988, Table 1). In K. elegans the presence of ovules maphrodite to imperfect owers (Ainsworth, 2000; Mitchell and Diggle, fl situated in the angle and a complete septum (i.e. below and above the 2005). In both oral morphs of K. elegans subsp. formosana the re- ovule insertion) separating locules within the ovary confirms the pla- productive organs develop in a similar way at early stages. However, centation as axile. A recent study reported axile placentation in K. bi- the subsequent developmental arrest of the opposite sex determines fl fl pinnata Franch. var. integrifoliola (Merr.) T.C. Chen (Cao et al., 2018). functionally imperfect owers. In functionally staminate owers, the Wang et al. (2013) recorded that K. bipinnata can eventually develop gynoecium is rudimentary and ovules abort, whereas in the functionally fl complete septa above the seed-bearing placental sutures. Nevertheless, pistillate owers the stamens are short, with anthers retaining their tetrasporangiate stage and containing empty, malformed, and not

105 ..Aao,e al. et Avalos, A.A.

Table 1 Comparison of selected characters of Koelreuteria elegans subsp. formosana with the available information for Xanthoceratoideae, Hippocastanoideae, Dodonaeoideae, Sapindoideae, and outgroups.

Taxa (Family/Subfamily/Species) No. ovules Curvature of mature ovule Micropyle Obturator Hypostase No. of nuclei in tapetal Pollen type References per carpel cells

Anacardiaceae 1 Anatropous Endostomic or Present or Present or 2 Tricolporate Copeland (1959); Burseraceae 2 Anatropous or campylotropous amphistomic absent absent 2 Tricolporate or Von Teichman (1988); Grundwag (1976); Kirkiaceae 1 Campylotropous Endostomic Absent – – tricolpate Bachelier and Endress (2009) Sapindaceae 5-8 Campylotropous Amphistomic Absent – 2 Tricolporate Narayana (1960); Harley et al. (2005); Xanthoceratoideae Amphistomic Absent Present Tricolporate Bachelier and Endress (2009) Xanthoceras sorbifolium Bachelier and Endress (2008) Acevedo-Rodríguez et al. (2011); Zhou and Liu (2012) Hippocastanoideae 2 Anatropous Endostomic Present Present 2, 3, or multinucleate Tricolporate Khuslani, (1963) Acer oblongum A. saccharum 2 Anatropous Endostomic Present Present 2 or multinucleate – Peck and Lersten (1991); Jacobs and Lersten (1994) Handeliodendron bodinieri 2 Campylotropous Amphistomic Absent Absent 1 Tricolporate Cao et al. (2008); Acevedo-Rodríguez et al. (2011) Dodonaeoideae Magonia pubescens 6-8 Campylotropous Endostomic Present Present 1 or 2 Trizono-colporate. González et al. (2017)

106 Calymmate tetrads Dodonaea viscosa 2 Anatropous Endostomic Present Present 2, 3, or multinucleate Tricolporate Karkare-Khushalani and Mulay (1964); Anzótegui and Ferrucci (1998) Filicium decipiens 1 Anatropous Amphistomic Present Absent 1 or 2 Tricolporate Gulati and Mathur (1981); Acevedo- Rodríguez et al. (2011) Sapindoideae 2 Campylotropous Amphistomic, zig-zag Present Absent 2 Tricolporate This paper Koelreuteria elegans subsp. formosana Pometia pinnata 1 Campylotropous Endostomic Present – 1 or 2 Tricolporate Ha et al. (1988) Nephelium lappaceum 1 Anacampylotropous Endostomic Present – 1 or 2 Tricolporate Ha et al. (1988) Xerospermum intermedium 1 Anatropous Endostomic Present – 1 or 2 Tricolporate Ha et al. (1988); Van der Ham (1990) Lepidopetalum jackianum 1 Anatropous Amphistomic Present – Multinucleate Tricolporate Mathur and Gulati (1981) Melicoccus lepidopetalus 1 Anatropous Endostomic Present Present 2 Tricolporate Zini et al. (2012) Allophylus alnifolius 1 Hemianatropous Endostomic Present Present 1 or 2 Triporate Mathur and Gulati (1980); Van der Ham (1990) A. edulis 1 Hemianatropous Endostomic Present Absent 1 or 2 Triporate González et al. (2014) A. zeylanicus 1 Anatropous – Present Present 3 Triporate Mathur and Gulati (1989); Van der Ham (1990) Urvillea chacoensis 1 ––––1 Hemitrisyn-colporate Solís et al. (2010) Cardiospermum halicacabum 1 Campylotropous Endostomic Present Present 1 Hemitrisyn-colporate Nair and Joseph (1960); Ferrucci and Anzótegui (1993) C. grandiflorum 1 ––––1 Hemitrisyn-colporate Solís et al. (2010); Ferrucci and Anzótegui (1993) Flora 255(2019)98–109 A.A. Avalos, et al. Flora 255 (2019) 98–109 viable pollen grains. Similar developmental pattern has been reported simultaneous cytokinesis, tricolporate pollen grain, unicellular stig- for other imperfect flower of Sapindaceae. (Nair and Joseph, 1960; matic papillae, bitegmic and crassinucellate ovules, Polygonum type Mathur and Gulati, 1980, 1981, 1989; Ha et al., 1988; Van der Ham, megagametophyte, and ephemeral antipodals are common features in 1990; Ferrucci, 1991; Cao et al., 2008; Solís et al., 2010; Acevedo- Sapindaceae. Additional comparisons with the designated outgroups Rodríguez et al., 2011; Vary et al., 2011; Zhou and Liu, 2012; Zini et al., Anacardiaceae, Burseraceae and Kirkiaceae suggest that these char- 2012; González et al., 2014, 2017) as well as in Aceraceae (Jacobs and acters may be plesiomorphic for Sapindaceae s.l. Reproductive char- Lersten, 1994; Hu et al., 2009; Yadav et al., 2016), Meliaceae (Styles, acters have not previously been subjected to ancestral state re- 1972; Gouvêa et al., 2008a), several representatives of Anacardiaceae constructions, therefore preliminary interpretations were provided in and Burseraceae (Wannan and Quin, 1991; Bachelier and Endress, this study. The results of the analysis did not resolve the ancestral state 2009), and Kirkiaceae (Bachelier and Endress, 2008). for the number of ovules per locule in Sapindaceae. However, an update All the anther tissues were well-differentiated in the stamens of of the frequency of the three possible character states evidences the functionally pistillate flowers of K. elegans subsp. formosana. However, prevalence of two ovules in the basal subfamilies Hippocastanoideae the middle layers and tapetal cells persist until anther maturity, and (100% of genera) and Dodonaeoideae (63%); whereas in Sapindoideae unviable pollen grains are concomitantly developed. Abnormal cy- predominates one ovule (93%). According to these values, it is inferred tology and delayed disintegration of the tapetum were previously re- that the presence of two ovules per locule would be plesiomorphic for lated with deviant pollen production in fully mature anthers of pistillate the family. Considering the 75 genera included in Sapindoideae, flowers of Cardiospermum grandiflorum Sw., Urvillea chacoensis Hunz. Koelreuteria shares two ovules per locule only with Ungnadia Endl., (Solís et al., 2010) and Magonia pubescens A. St.-Hil. (González et al., Delavaya, Erythrophysa E. Mey. ex Arn., and Conchopetalum Radlk. 2017). Moreover, the persistence of both tapetal cells and middle layers Nevertheless, the presence of an obturator, which was reconstructed for were linked with the formation of abnormal cytoplasm and sporoderm the common ancestor of the clade Dodonaeoideae and Sapindoideae, in the microspores of Melicoccus lepidopetalus Radlk. (Zini et al., 2012), suggests that Koelreuteria may be morphologically related with either of and with the presence of malformed pollen grains in Allophylus edulis the two subfamilies. On the other hand, the evolutionary analysis re- (A. St.-Hil.) Niederl. (González et al., 2014). vealed binucleate tapetal cells as plesiomorphic for Sapindaceae. The The absence of anther dehiscence is another crucial mechanism character may thus be of phylogenetic significance for higher level involved in androecial sterility in structurally hermaphrodite flowers of clades, although more information on the tapetum, at least for Sapindaceae. Although an endothecium with secondary fibrous thick- Dodonaeoideae, Hippocastanoideae, Sapindoideae and the family enings is a requisite for the mechanical dehiscence process of the anther Kirkiaceae, is needed due to the existence of character polymorphism in in plants (Keijzer, 1987), in both floral morphs of K. elegans subsp. terminal taxa. Similarly, it is necessary to reevaluate the ovule curva- formosana the endothecium developed in the same way. Since breakage ture and micropyle type in which ancestral states are equivocal. These of the intersporangial septum and stomium is necessary in the anther results indicate that parsimony reconstruction of ancestral states could dehiscence process (Goldberg et al., 1993; Beals and Goldberg, 1997; be helpful to morphologically characterize clades with molecular sup- Yang and Zu, 2016), a defect in the programmed degradation of both port at familial and subfamilial levels. Future studies in Sapindoideae tissues contributing to preventing pollen release in hermaphrodite taxa, the most abundant subfamily, are necessary, and they should be flowers is suggested. In other Sapindaceae, this event is accompanied by directed to further evaluate the phylogenetic significance of these the presence of low lignification of the endothecium cells, as occurs in characters including new character sets that have not been examined C. grandiflorum and U. chacoensis (Solís et al., 2010), fibrous thickenings yet, such as carpel number, stigma surface (unicellular vs. multi- only in three or five cells as in M. pubescens A. St.-Hil. (González et al., cellular), style type, and orbicules. 2017) or a complete lack of cellular radial enlargement and wall thickenings, in M. lepidopetalus and A. edulis (Zini et al., 2012; González Acknowledgments et al., 2014). 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