The Significance of Fruit and Seed Anatomy in the Evolution of Sabiaceae

THE SIGNIFICANCE OF FRUIT AND SEED ANATOMY IN THE EVOLUTION OF SABIACEAE

Ludovica Santilli 2016 Thesis submitted in partial fulfillment for the MSc in Biodiversity and Taxonomy of Plants

Abstract

Fruit and seed morphology and anatomy of selected species of Ophiocaryon and Meliosma were investigated to gain insight into the evolution of fruit and seed related characters in the Sabiaceae and to find possible distinctive characters for the family in relation to their position at the base of Proteales. The investigation implied the experimentation of three different embedding techniques, the optimisation of the protocols with regards to the material object of this study, and the use of microtomy and of light microscopy. Carpological characters are often indicators of taxonomic affinities and used to distinguish taxa at different level. Similarly, seed morphology and anatomy, especially when the seed is investigated in the context of its development -from its early embryonal phase to its maturity- are considered important source of phylogenetic relationships. The fruit of Sabiaceae has developed into a drupe in which the pericarp becomes hard and takes over the seed protection, while the seed coat is reduced and partly disintegrated or disappears during development. Some species of Sabiaceae have ovules that have become unitegmic probably by loss of the outer integument and are hemitropous to orthotropous. These two derived conditions from an ancestral bitegmic and anatropous ovule probably go together as the outer integument, is thought to be responsible for curvature of ovules in angiosperms. The ovules develop into orthotropous exalbuminous seeds with a collapsed and undifferentiated seed coat. The orthotropous and pachychalazal seed of Ophiocaryon might be derived from the orthotropous seed of Meliosma which does not show pachychalazaly. As for the possible characters shared with other early diverging eudicots, this study could not find unequivocal evidences. Nevertheless, the most interesting features to be further investigated in order to address the question have been identified. Additional, anatomical investigation on ovules and seeds of Sabiaceae would confirm the process through which unitegmy has been achieved and allow direct comparison with those members of Proteales that show orthotropy and reduction of the outer integument.

Acknowledgements

I wish to thank my supervisors Wolfgang Stuppy and Louis Ronse de Craene for their support throughout the project.

Lab work was a delicate, time consuming part of the project. Sincere thanks go to Frieda Christie for her assistance and precious advices during the lab work, which allowed me to improve the protocol and to get beautiful and clear LM images. I would also like to thank Michael Muller for finding the time to answer my questions on histochemical tests and staining procedure and Greg Kenicer for his help in the moment of need.

Most of all, I want to express my gratitude to my friends. My classmates for being such a good, unforgettable team, particularly Camila for cycling together to the garden, Jia my darling for your sweetness, Marita for letting me complain the all year and Nicolas for all the time together and all the little things. Roberta, for spending the nights writing in the library of the MSB and make it incredibly fun. Chiara and Fruszi, for understanding everything I say and even more.

Finally, to my family. My mother who made this possible, my sister who will be a great doctor and my little brother who has inspired me with his courage, I am very proud of you both.

Table of contents

ABSTRACT ...... 1 ACKNOWLEDGEMENTS ...... 2 TABLE OF CONTENTS ...... 3 TABLE OF FIGURES ...... 4 1 INTRODUCTION ...... 5

1.1 GENERA OF SABIACEAE ...... 5 1.2 TAXONOMY...... 6 1.2.1 Phylogenetic position of Sabiaceae ...... 6 1.2.2 Infrageneric classification ...... 8 1.3 MORPHOLOGY ...... 10 1.3.1 Floral morphology ...... 10 1.3.2 Gynoecium ...... 11 1.3.3 Fruit and seed morphology ...... 12 1.4 AIMS OF THE INVESTIGATION ...... 13 2 MATERIALS AND METHODS ...... 14

2.1 PLANT MATERIAL ...... 14 2.2 EMBEDDING WITH TECHNOVIT ...... 14 2.3 EMBEDDING WITH LR WHITE RESIN ...... 16 2.4 CRYOSECTIONING ...... 17 2.5 HISTOCHEMICAL TESTS ...... 18 2.6 LIGHT MICROSCOPY ...... 18 2.7 TERMINOLOGY USED ...... 18 3 RESULTS ...... 19

3.1 FRUIT DEVELOPMENT ...... 19 3.1.1 Ovary wall ...... 20 3.1.2 Pericarp ...... 22 3.2 SEED DEVELOPMENT ...... 27 4 DISCUSSION ...... 31

4.1 DIVERSIFICATION WITHIN SABIACEAE...... 31 4.2 EVOLUTIONARY TRENDS IN SABIACEAE ...... 32 4.2.1 Fruit development and evolution ...... 32 4.2.2 Seed form and integuments ...... 34 4.3 COMPARISON WITH CLOSEST FAMILIES...... 36 CONCLUSIONS ...... 40 REFERENCES ...... 42 APPENDIX ...... 48

Table of figures

Cover page Meliosma bogotana Steyerm. Photo by John Bernal, Jardin Botanico de Bogotà. Figure 1 Phylogeny of early diverging Eudicots ...... 7 Figure 2 Phylogeny of Sabiaceae...... 9 Figure 3 A-D= M. cuneifolia gynoecium at anthesis E-F= O. heterophyllum young fruits. A= LS along the frontal plane B= LS details of pericarp layers and ovule. C= TS showing carpels anatomy and ovules D= TS Details of pericarp layers and ovule. E= LS along the median plane F= LS along the median plane ...... 21 Figure 4 O. heterophyllum. A,C= LS of young fruit B,D= TS of fruit at the same stage of development...... 23 Figure 5 M. cuneifolia. LS of young fruits showing pericarp anatomy. A= Carpels equally developed B= The unequal development of the carpels changes the original orientation of the pericarp and seed. C= Later stage D= detail of the subepidermis and of the endocarp E= Close up of the exocarp F= Close up of the exocarp...... 25 Figure 6 M. cuneifolia LS of old stage. A= Well differentiated B= Close up of the exocarp C= Same details of a slightly younger stage D= Detail of the vascular bundle and crystal E= Detail of the sclereids ...... 26 Figure 7 O. heterophyllum old stages. A= TS of older stage but not mature fruit. B= LS showing details of mesocarp and endocarp. C= LS close up on micropylar end of the seed...... 28 Figure 8 M. cuneifolia close up of the vestigial seed coat...... 29 Figure 9 A,C,D= O. maguirei B= O.eterophyllum C= Cryosection of the embryo D= Endocarp. E= Sabia paniculata. F= Meliosma alba...... 30 Table 1 Comparison of Sabiaceae with other early diverging Eudicots on a number of selected characters...... 39 Continuation of Table 1 ...... 40

1 Introduction

1.1 Genera of Sabiaceae

Sabiaceae Blume is a small family comprising three genera of evergreen, rarely deciduous trees, shrubs and woody lianas with spirally arranged, simple or imparipinnate leaves (Kubitzki, 2007). The largest genus of the family is Meliosma Blume with Southeast Asian and tropical American distribution, where it occurs mostly south of Central America and in the tropical Andes (Lombardi, 2009). The most recent taxonomic revision of Meliosma was done by Van Beusekom (1971) who deals exclusively with the SE. Asian sections of Meliosma and does not treat the American section Lorenzanea which occurs only in the New World. He recognised only 15 of the 100 estimated Asian species of Meliosma. With regards to Neotropical Meliosma, Urban (1900) described 17 species from Mexico, Central America, the Caribbean islands, Colombia and Brazil. Since then, 69 (Ramos, 2012) new Meliosma species from the Americas have been described by many authors (Gentry, 1992; Arbeláez 2004; Cornejo 2008; Cornejo, 2009; Lombardi, 2009). Meliosma includes evergreen, rarely deciduous trees with simple or imparipinnate leaves and many-flowered terminal or axillary panicles. Flowers are bisexual, actinomorphic with five petals, the inner two strongly reduced. The androecium comprises two polliniferous and three sterile stamens and the gynoecium is bicarpellate (Kubitzki, 2007). Sabia Colebrooke, was most recently revised by Van de Water (1980) who reduced the species known at that time from 55 to 19 and described the genus as mainly confined to SE. Asia and the Malesian. About 30 species have recently been reported (Guo and Brach, 2007). Sabia comprises evergreen or deciduous lianas and scandent shrubs with simple leaves. It is differentiated from the other two genera mainly in having actinomorphic flowers, solitary or in axillary panicles with all five stamens polliniferous (Kubitzki, 2007). The third genus of the family, Ophiocaryon Endlicher, is found exclusively in the new world in moist forests of Colombia, Venezuela, Guyana, Ecuador, Peru, and Brazil, especially on the Guayana Shield and in the Amazon basin (Aymard and Daly, 2006). This is the smallest genus of the family with nine species reported by the last authors. Before Aymard and Daly described the two new species, the only other taxonomic revision available in literature was the one by Barneby (1972) for the Flora of Guyana. Barneby

(1972) divided the seven species of Ophiocaryon known at the time, into two series Phoxantus and Ophiocaryon based on petal shape character. Ophiocaryon includes evergreen small trees with simple or imparipinnate leaves. The flowers are more similar to Meliosma than they are to Sabia with the difference that the petals are not so dimorphic (Kubitzki, 2007).

1.2 Taxonomy

Some authors did not support the homogeneity of the family and separated the genera into the two distinct families based on the observation that the fruit and flower of Ophiocaryon and Meliosma share more similarities that they do with Sabia (Dahlgren, 1981; Takthajan, 1997). Takhtajan (2009) consecutively placed Sabiaceae in its own order, Sabiales and separated Meliosma and Ophiocaryon from Sabia, treating them respectively in Meliosmoideae and Sabioideae. Most recent authors treat the three genera in the single family Sabiaceae (Kubitzki, 2007; Stevens, 2001 onwards).

1.2.1 Phylogenetic position of Sabiaceae

Regarding its relationships with other families, Sabiaceae has been in a pre-molecular age associated, even though with some uncertainty, with Sapindaceae (Bentham, 1862; Dalhgreen, 1980), Ranunculales (Cronquist, (1981), or Rutales (Carlquist, 1993). Johri et al. (1992), considered the association of Sabiaceae with Sapindales inappropriate based on ovule-related characters and the exclusion of the family from Sapindales has been supported by molecular studies (eg. Gadek, 1996). Phylogenetic studies based on molecular data such as plastid genes rbcL (Chase, 1993; Gadek, 1996; Savolainen, 2000a), matK (Hilu et al., 2003, 2008), combined plastid genes (Savolainen, 2000b; Worberg et al., 2007; Moore et al., 2010; Barniske et al., 2012; Sun, 2016) combined plastid, mitochondrial and nuclear (Soltis et al., 2000, 2003, 2011; Kim et al., 2004; Qiu et al., 2005) genes, have consistently placed Sabiaceae within the early diverging eudicots. These studies sampled both widely across angiosperms, as well as among early diverging eudicots specifically. Sampling of Sabiaceae for phylogenetic

Figure 1 Phylogram of the best tree determined by RAxML for the 79-gene, 97-taxon data set using 34-partition scheme (ln L = 1108895.169560). Numbers associated with branches are ML bootstrap support values; asterisks indicate ML BS = 100%. The range of ML bootstrap values for all other schemes (including single partition, 2-partition, 3- partition, 79- partition, 158-partition and 237 partition) are presented in parentheses; the presence of a single number indicates all analyses shared the same support value.

8 analyses has never included Ophiocaryon species. Nevertheless, Sabia and Meliosma always form a clade and none of these studies rejected the monophyly of the family. Despite the agreement of these studies in placing Sabiaceae among the basal branches of eudicots, its exact position relative to the other families is still unresolved. The sister relationship Sabiaceae-Proteales was resolved with the highest support (bootstrap >90% in ML) in the plastid phylogenetic analysis by Sun et al. (2016) while it received low to moderate statistical confidence in previous studies where Sabiaceae appeared alternatively branching immediately after Ranunculales or as sister to Proteales (Hilu et al., 2003, Soltis et al., 2003, 2011; Qiu et al., 2005; Worberg et al., 2007; Moore et al., 2010; Barniske et al., 2012). Kim et al. (2004) suggested a position of Sabiaceae near Trochodendrales or Buxales but, given the results of the majority of the other studies, this scenario seems improbable. Worberg et al. (2007) pointed out that Takhtajan’s (1997) attempt to place Sabiaceae in its own order Sabiales, was difficult to apply due to the fact that a possible sister-group relationship of Sabiaceae to Proteales could not be excluded. Hence the option of including Sabiaceae into Proteales. This last scenario is the currently accepted by the majority of authors (Stevens, 2016; APG IV, 2016). The fact that Sabiaceae share more morphological characters with Ranunculales (such as Menispermiaceae) than with Proteales (see Ronse De Craene and Wanntorp, 2008; Ronse De Craene et al., 2015a,b) makes a definite answer difficult and supports a need for investigating clear synapomorphies linking Sabiaceae with Proteales.

1.2.2 Infrageneric classification

With regards to the relationships within the family, the only phylogenetic study based on molecular evidences was done by Zúñiga (2015). The author aimed to reconstruct the backbone of phylogenetic relationships within the family using sequences of slowly evolving plastid loci (rbcL and trnL-F), and evaluate species-level relationships within Meliosma by means of more rapidly evolving nuclear and plastid loci (ITS, psbA-trnH, and rpl32-trnL). A total of 65 species of Sabiaceae were sequenced. The outgroups represented three accessions from Ranunculaceae Juss. and Proteaceae Juss. The genus Meliosma was

9 sampled including representatives of the all infrageneric taxa recognised at the time (mostly based

Figure 2 Majority-rule consensus tree from Bayesian inference analysis on supermatrix including five loci and 85 taxa (recorded presence/absence indel data included). Posterior probabilities (bold, on all clades) and maximum likelihood bootstrap support values shown above branches, parsimony bootstrap shown below branches. ML and parsimony bootstrap values shown only on well-supported clades (≥ 70%). Geographic locality of sample indicated when relevant. Infrageneric classification according to Van Beusekom (1971). Branch lengths are shown for one of best trees found on ML analyses of the super-matrix (inserted in upper left corner). Abbreviations for genera: A. = Aquilegia, G. = Grevillea, M. = Meliosma, O. = Ophiocaryon, P. = Platanus, S. = Sabia.

10 on Van Beusekom classification, 1971) and from the all distribution range. Sabia was sampled including as many species as possible while Ophiocaryon included members of the two series designated by Barneby (1972) Phoxanthus and Ophiocaryon. The author underlines the impossibility of testing the monophyly of these two series due to the unavailability of adequate number of specimens and poor quality of DNA extracted. Results showed Sabia diverging first within the family. Surprisingly, despite the support for the monophyly of Sabia and Ophiocaryon, Ophiocaryon is nested within Meliosma in analyses including chloroplast data. Specifically, Ophiocaryon appears to be sister of Meliosma alba Schltdl., placing doubts on the monophyly of Meliosma. As the author highlights, the presence of the explosive pollen release mechanism of Meliosma has been questioned in M. alba (Ronse De Craene and Wanntorp, 2008), and M. alba was considered the least derived species of the genus by Van Beusekom (1971). Given the morphological similarities, which well justify a distinction between Meliosma and Ophiocaryon, the author suggests to transfer M. alba to a new genus rather than including Ophiocaryon in Meliosma. The author also discusses Van Beusekom’s (1971) infrageneric classification of Meliosma based on the tree topology and attempt to hypothesise the endocarp evolution in Meliosma. The findings provide only partial support for taxa recognized by Van Beusekom (1971), suggesting that some infrageneric taxa are not monophyletic.

1.3 Morphology

1.3.1 Floral morphology

Sabiaceae are unique among members of the eudicot grade in having the perianth differentiated into a calyx and corolla and a nectary disc (Stevens, 2016). Among early- diverging eudicots, nectary discs are found also and only in Buxaceae Dumort. and Proteaceae. (Kubitzki, 2007). Characters shared by all members of Sabiaceae are: basically pentamerous flowers (with some tetramerous exceptions in Ophiocaryon), disporangiate stamens opposite the petals, superior, bicarpellate ovary with two ovules each carpel, a nectary disc and drupaceous fruits in which usually only one seed develops (Barneby, 1972; Kubitzki, 2007; Wanntorp and Ronse De Craene, 2007).

Recent investigations on floral morphology and floral development have shed light on details of flower morphology and flower evolution of the two genera Sabia and Meliosma (Ronse De Craene et al., 2015a, 2015b; Wanntorp and Ronse De Craene, 2007; Ronse De Craene and Wanntorp, 2008). These and other studies (Soltis et al., 2003), show evidences of a unique and independent origin of pentamery from a possible trimerous progenitor and point that the floral morphology of Meliosma and Sabia is comparable supporting their treatment within the single family Sabiaceae. There are many morphological characters supporting the close relationship Ophiocaryon- Meliosma that lead many authors in segregating them in Meliosmaceae and considering a monogeneric Sabiaceae. Flowers are actinomorphic in Sabia to strongly zygomorphic and extremely complex in Meliosma. Ophiocaryon flowers are more similar to Meliosma in having monosymmetric flowers due to reduction in size of two petals and a partially sterile androecium, in contrast to Sabia (Ronse De Craene et al., 2015b). Sabia is also the only genus in the family that comprises woody climbers as well as shrubs and small trees. The base of the ovary is surrounded by a conspicuous nectary with five prominent appendages alternating with the stamens and staminodes.

1.3.2 Gynoecium

The ovary is superior and syncarpous with two carpels corresponding to two locules each containing two superposed ovules. The placentation is lateral, marginal or axile. Ovules have been described as hemianatropous, unitegmic and crassinucellar (Raju, 1952; Van de Water, 1980; Johri et al., 1992; Endress and Igersheim, 1999). However, Ronse De Craene and Wanntorp (2008), found that M. veitchiorum Hemsl. showed two integuments with the outer one occasionally reduced, and that some selected species of both Sabia and Meliosma are orthotropous or slightly curved with short funiculus. Orthotropous ovules were described also by Endress and Igersheim (1999) as a shared feature between Sabiaceae, Proteaceae, and Platanaceae T.Lestib. Endress and Igersheim (1999) describe the single integument as annular, not convoluted and six-cell-layers thick. The absence of a micropyle reported by Raju (1952) has not been contradicted by later studies. Of the usual four ovules, generally only one develops into a seed (Van Beusekom, 1971; Van de Water, 1980, De Craene et al., 2015b).

1.3.3 Fruit and seed morphology

Ophiocaryon and Meliosma share a subglobose drupe with a thin, fleshy or completely dry mesocarp and a prominent keel running around the thin-walled, smooth-surfaced endocarp (Kubitzki, 2007; Zúñiga, 2015). In Meliosma species, the vascular bundle connecting pedicel and seed can be situated either outside or inside the endocarp wall (Van Beusekom, 1971). At maturity, the endosperm is reduced or absent and the embryo has developed cotyledons on an extended, curled hypocotyl (Kubitzki, 2007) Sabia can produce both solitary and schizocarpic fruits as a pair of single-seeded endocarps developing per flower. In the last case, two basally connected drupelets per flower, with the persistent style situated between them, are produced. More often, one of the two locules is not developed and when only one drupelet is formed, the persistent style is situated at its base (Van de Water, 1980). The mesocarps are fleshy and thin. The endocarps have a reniform shape, lenticular in cross section, and are sclerenchymatous and reticulately ridged, very often with a more or less prominent rib. The seeds are characterized by a conspicuously dark-dotted testa and the endosperm is essentially reduced (Raju, 1952; Van de Water, 1980; Kubitzki, 2007; Manchester and Kodrul, 2014). The mature embryo of Sabia has been described by Raju (1952) as having the hypocotyledonary region twisted, and by Van de Water (1980) “with two flat, smooth, somewhat undulated, or sometimes strongly folded cotyledons and a cylindrical rootlet curving to the hilum”. Fruit and seed anatomy of the family, especially of Ophiocaryon is poorly known.

1.4 Aims of the investigation

Fruit and seed anatomy is considered to be of taxonomic and phylogenetic relevance. The structure of the seed coat for instance is characteristic in many families or groups of families and possibly below the family level (Bouman, 1978). Testa structure for example may support taxonomic distinction at the family and generic levels as in Malvaceae Juss., Bombacaceae Kunth, Cucurbitaceae Juss., Fabaceae Lindl. and Brassicaceae Burnett (Vaughan, 1968). Ontogenetic studies are important when attempting to establish relationships because they allow us to establish whether the mature structures of different taxa are homologous- and thus comparable- or have different origin. The aims of this study are to investigate the fruit and seed anatomy of one species of Ophiocaryon and one species of Meliosma and compare their development. Particularly, the interest is to gain insight into the evolution of integuments in Sabiaceae and find distinctive seed and fruit characters for the family in relation to their position at the base of Proteales.

2 Materials and methods

2.1 Plant material

Young fruits of Meliosma cuneifolia Franch (accession number 19381038) in cultivation at the Royal Botanic Garden Edinburgh (RBGE), were collected, stored in 70% ethanol (EtOH) and embedded with both Technovit and LR Resin (see below). Young fruits of Ophiocaryon heterophyllum (Benth.) Urb. were collected by Euridice Honorio in plots in Peru from different trees (tree codes: 5C 1/151; 2K 7/180; 4A 1/85). Stored in 70% EtOH, this material was embedded with Technovit. Fruits of the following specimens were collected from herbarium material and used for macromorphological observations and cryosectioning: Meliosma alba (Schltdl.) Walp. (Forrest 27336, Asia, RBGE), Meliosma dentata, (Liebm.) Urb. (C.G. Pringle 4371, Central America RBGE), Meliosma pinnata (Roxb.) Maxim ssp. macrophylla (Merr.) Beusekom (Takeuchi 11190, Asia, RBGE), Ophiocaryon paradoxum R.H. Schomb (180, KEW), Ophiocaryon paradoxum (1881 KEW), Ophiocaryon manauense (W.A.Rodrigues) Barneby (Pennington 17013, Peru, KEW), Sabia paniculata Edgew. ex Hook. f. & Thomson (JF Doremez 2437, Asia, RBGE). Fruits of all herbarium specimens were photographed and the measurements of length, width and height were taken. Fruit of all pickled material of big size was scarified prior to dehydration to favour the infiltration of the resin. That is, cutting a section from the fruits (parallel or perpendicular to the longitudinal axis) with a razor blade.

2.2 Embedding with Technovit

Specimen preparation for compound light microscopy required the following processes: dehydration; embedding; cutting; mounting and staining. Dehydration involved the gradual removal of water from the tissue through a series of graded EtOH solutions. Dehydration was performed using 90% and 96% EtOH. The duration of each step was 2 hr during which the material was placed into a vacuum. Finally the material was transferred into 100% EtOH and left overnight. After this, the tissue was infiltrated with the hydroxyethylmethacrylate based resin Technovit 7 100 (Heraeaus Kulzer, Wehrheim, Germany). The Infiltration Medium (IM)

15 was prepared with 100 ml of Technovit 7100 liquid for every 1g of hardener I (dibenzoylperoxide) and kept refrigerated all time. The objects were transferred into a series of solutions of absolute EtOH and IM in proportion 5:1, 3:1, 2:3, 1:5. The tissue was infiltrated gradually using these solutions in sequence (2 hr each). To facilitate the infiltration, the samples were placed in the vacuum at each step and then transferred to 100% IM and left overnight at room temperature. The whole infiltration process was performed without agitating the tissue. Thereafter, the material was transferred into the embedding medium (EM) in the Technovit histoform S embedding molds. The EM was prepared with IM and hardener II in proportion 11:1. In order to prevent premature polymerization, both IM and hardener II were kept refrigerated prior to embedding. The histoform S embedding mould was also kept in the fridge for the same reason. The EM was poured into the holes in the histoform and the holes were completely filled. Each object was then placed in the middle of each hole using forceps and, oriented with the longitudinal axis parallel to the longest side of the hole (parallel to the bottom of the hole). Occasionally, when two young fruits of the same size were present, one was oriented perpendicular to the bottom of the hole in order to obtain cross sections during cutting. As soon as the hardener II and the IM are mixed, the fluid starts hardening, but for some time the mixture is not viscous enough to prevent objects movements. Thus, samples were controlled regularly and the orientation adjusted for the following 30 minutes. After this time, the fluid was so viscous that the objects could not be rearranged anymore. When the objects were floating in the EM as a sign of presence of air inside the tissues, they were placed back in 100% IM to progress the infiltration. The histoform base was placed on the hot plate set at 40°C for 1 hour and left overnight at room temperature. Prior to mounting, the surface of each resin block was dried with paper. The white histoblock holders (Heraeaus Kulzer) were labelled to identify each sample and inverted over the corresponding sample. The mounting medium was prepared with Technovit 3040 yellow (Heraeaus Kulzer), a fast curing methyl methacrylate-based resin. The two components powder and liquid (refrigerated), were combined in proportion 2:1, well mixed, and rapidly poured into the holes of the histoblock holders. After 10 minutes

16 the resin solidified and the blocks could be removed with the block holders from the mould. The specimens were trimmed with a razor blade and re-trimmed during cutting if necessary. The specimens were cut with a Leica RM 2235 Microtome using the knife holder E-TC and Leica TC65 tungsten carbide disposable blades. Sections width ranged between 3 and 10 μm according to the different specimens and grade of infiltration of the resin. In order to keep the sections attached to the slides during the staining process, paratissuer (FEEC) was applied on the slides, finally they were covered with a layer of distillate water

(dH2O) and brought onto the hotplate at 45°C. Sections were individually collected from the knife holder and placed onto the slides. When the water evaporated the slides were left air drying until staining. For the staining, a solution of 0.02% of Toluidine blue was prepared with 0.02 g toluidine blue and 100 ml of dH2O. The sections were placed into a section holder and brought into a sequence of baths. In order: Toluidine blue (2 min), dH2O (few seconds), 70% EtOH (1 min), 95% EtOH (1 min), absolute EtOH 1 min, Histoclear, a histological clearing agent (3 min). Once removed from the clearing agent, the lower surface of the slides was dried with paper. Cover slips were laid down and a generous amount of DPX Mountant, a mixture of distyrene, a plasticizer, dissolved in toluene-xylene was linearly spread by length with the use of a glass pipette. The slides were then inverted on the cover slips and after a few seconds, when the DPX reached the borders of the coverslips, they were flipped back. Excess of DPX was removed with a razor blade and the slides were left air drying overnight.

2.3 Embedding with LR white resin

Fruits embedded with this procedure, required first to be dehydrated through a series of graded EtOH solutions. Dehydration was performed using 80%, 90% and absolute EtOH. The duration of each step was 24 hr. The resin used for this embedding was a hydrophilic acrylic resin of low viscosity (8 cps) and medium grade (LR white resin). The material was transferred from absolute EtOH to a

17 series of solutions of resin and absolute EtOH. The proportions were in the following order: 1:2, 1:1, new 1:1, 2:1, 1:0, new 1:0. The duration of each step was 24 hours. Specimens were transferred individually into gelatin capsules supported by the edges of holes cut into the lids of TEM film boxes, for polymerisation. The fruits were oriented with the longitudinal axis parallel to the bottom of the capsules. The capsules were filled for less than half of their length with the resin and the lids of the capsules were not placed back. The boxes were left for 18 h in the vacuum oven (Binder) at 60°C at 440mmHg to allow polymerisation. The boxes were then placed in the fume cupboard to allow the resin to harden. Gelatin capsules were then dissolved in warm water, dried with paper and ready for cutting. The material was sectioned with an automated microtome (Reichert-Jung, Mod. 1140/autocut). The slides were prepared at the same way as the ones aforementioned. To stain the sections, toluidine blue was poured on the slides with a pipette and after a few seconds the slides were rinsed with dH2O. Left overnight to dry, the slides were permanently fixed as before the following day.

2.4 Cryosectioning

Fruits of M. alba, M. dentata and seed of O. maguirei were soaked in tap water for 24 hr to soften the tissues. A very thin layer Kaiser’s glycerol-gelatin was spread on each slide in order to make it sticky. To bond and encapsulate tissue specimens to the object holder for cryosectioning, the fruits were embedded in a water soluble embedding compound for frozen sectioning (FSC 22 clear, Leica). The fruits were oriented by this time in the embedding medium in order to get longitudinal sections. The cutting process was carried out at a working temperature of -20 °C. Sections were taken at different width between 10 and 20 μm. Each fruit was sectioned until slightly beyond the middle and the left overs were placed in 70% EtOH for conservation. Seed coat remains of O. maguirei were placed in 100% EtOH. Sections were left overnight in the microtome to frost. The next day the slides were placed on a hotplate to remove the water content and then let dry.

Under the fume cupboard the slides were prepared in the following way: first, they were sprayed with EtOH to remove any remain of water content. Once the EtOH evaporated, a few drops of Histoclear were poured on the sections. Next, a few drops of a quick-hardening mounting medium were poured on the sections (Eukitt, Agar Scientific). Coverslips were then placed on top of the slides and left overnight with weight on top.

2.5 Histochemical tests

The Iodine-Potassium-Iodide test (IKI test) was conducted on sections of M. cuneifolia and O. heterophyllum to detect the presence of starch. The IKI solution was prepared by first dissolving 2 g of KI in 100 ml of water, and adding 0.2 g of iodine into the KI solution. The solution was prepared 24 hr before use as iodine takes some time to dissolve. The solution was stored in a dark glass bottle well tight as exposure to light and air degrades the solution’s usefulness.

2.6 Light microscopy

Pictures were taken at different magnifications with Axiophot fluorescence microscope (Zeiss) and AxioVision software (AxioVs40 V 4.8.2.0).

2.7 Terminology used

The fruit-type classification and the terminology to define the pericarp layers was based on Roth (1977), the nomenclature used to describe the seeds was that of Corner (1976).

3 Results

The clearest sections were obtained with M. cuneifolia embedded with LR white resin procedure and O. heterophyllum embedded with Technovit resin procedure when a big section (almost half of the fruit) were cut off during scarification. Anatomical observations of the pericarp layers were possible with these two species only. All herbarium specimens of the other species of Meliosma did not contain seeds. Furthermore, these fruits were damaged and pressed and observation of these species with regard to shape and textures was not possible. Herbarium specimens of Ophiocaryon were mature and in good condition. Cryosectioning was possible on their seeds only but, as the fruits are relatively big, macromorphological observations on the pericarp and, on the seed itself were possible. The fruit in both genera is a drupe, which is a type of fruit characterised as an indehiscent sarcosclerocarpium with fleshy mesocarp and hard endocarp that generally encloses a single seed (Roth, 1977). Although in this study the actual sizes of the mature fruits could not be measured for Meliosma spp., due to the issues aforementioned, the fruits of Ophiocaryon spp. are certainly much bigger. The herbarium material available of Ophiocaryon shows drupes of subglobose shape and bilateral symmetry with smooth, leathery surfaces of the pericarp and reticulate pattern of the surface of the endocarp (Fig. 9A-D). All endocarps observed, showed a plane of dehiscence corresponding to the more or less prominent keel running along the plane of symmetry of the endocarp itself.

3.1 Fruit development

Ideally, this section should describe the anatomy of the fruits at well-defined stages, demarcated by the absolute age of the fruits. However, due to the lack of material and time constraints, observations throughout the fruits development were not continuous. Observations of the gynoecia of O. heterophyllum and M. cuneifolia at anthesis were made on anatomical sections provided respectively by Thaowetsuan (2016, unpublished) and Ronse De Craene and Wanntorp (2008) (Fig. 3A-E). For the last species mentioned, it was possible to observe the young fruits when the two carpels are equally developed (Fig. 3F, 4, 5) and when one of the two begins to stop its growth and the other prevail on the

20 development. Unfortunately, if in these stages it is possible to see the main features of pericarp development, the seed was always damaged and its development could not be observed. The oldest stages available of M. cuneifolia showed fruits with their major tissues differentiated and a clear embryo immersed in conspicuous endosperm (Fig. 6a). Dissimilarly, the oldest stage of fruits O. heterophyllum, embedded with resin, showed fruits whose endocarp is not yet completely lignified, the embryo not developed and endosperm abundant (Fig.7). Consequently, the events between this stage and the mature stage -as observed at macromorphological level on different species of Ophiocaryon- can only be hypothesised. From this, the decision to divide the description of the fruit development into the two sections of the anatomy of the ovary wall and of the pericarp, with information on the meristematic tissues involved in the origin of the different pericarp layers. Generally, the ovary of both Ophiocaryon and Meliosma is superior, bicarpellate, syncarpous and bilocular. The ovules are originally two in each locule and have marginal placentation (Fig. 5A). One of the carpels ceases its growth soon in development and, as the other increases in size, the developing ovule starts rotating and the region of the placenta will eventually end up close to the pedicel (Fig. 5B-C, 6A). Between this region and the pedicel, the aborted carpel with a very small locule and undifferentiated pericarp layers was frequently still visible. Of the two remaining ovules, only one developed into a seed.

3.1.1 Ovary wall

In both O. heterophyllum and M. cuneifolia the ovary wall is clearly differentiated into three zones during anthesis: outer epidermis, mesophyll, and inner epidermis (Fig. 3). The outer epidermis in both O. heterophyllum and M. cuneifolia is uniseriate, of isodiametric cells with thin, straight anticlinal walls and visible nucleus. These cells show anticlinal division. A hypodermis of isodiametric cells is present in both species. These cells differentiated from the ovarian mesophyll in cell content. In M. cuneifolia the hypodermis is mostly uni- or bi-seriate and the cells are of the same size of the outer epidermis cells. A few cells show periclinal division. In both O. heterophyllum and M. cuneifolia, the ovarian mesophyll is formed of several layers of large globose parenchymatous cells with evident nucleus.

Figure 3 A-D= M. cuneifolia gynoecium at anthesis from Ronse De Craene and Wanntorp, 2008). E-F= O. heterophyllum young fruits. E from Thaowetsowan (2016, unpublished). A= LS along the frontal plane showing ovary differentiation and two superposed ovules. B= LS details of pericarp layers and ovule. C= TS showing carpels anatomy and ovules with marginal placentation. D= TS Details of pericarp layers and ovule. Inner epidermis showing meristematic activity involved in endocarp formation while subepidermis remains one-layered. E= LS along the median plane showing ovary layers and two superposed ovules of one of the two carpels. F= LS along the median plane at the stage when the carpels are still equally developed. Oe=Outer epidermis, M=ovarian Mesophyll, Ie=Inner epidermis, Ov=Ovule, Es=Embryo sac, Red arrow=Hypodermis, White arrow=Subepidermis, Arrow head=Periclinal division.

The inner epidermis of the ovary of O. heterophyllum at anthesis could not be observed clearly. In M. cuneifolia is uniseriate but shows active anticlinal as well as periclinal division (Fig. 3D). This is a zone of meristematic activity that will give rise to the endocarp in later stages. Just above the inner epidermis a single layer of cells differentiate from the mesophyll. They present dense cytoplasmatic content and show anticlinal division only. This layer is present in both species and persists during development until the layers of cells of the endocarp underneath become completely lignified (Fig. 3, 4, 5A- D).

3.1.2 Pericarp

During fruit development, the pericarps of both O. heterophyllum and M. cuneifolia differentiate into three histological zones: exocarp, mesocarp, and endocarp developing correspondingly from outer epidermis, ovarian mesophyll and inner epidermis (Fig. 5C, 6A, 7A). The exocarp consists of an outer epidermis and a hypodermis. The outer epidermis is mostly uniseriate at the different stages of development observed and presents anticlinal division activity. Occasionally, a periclinal division was also detected, leading to the idea that the hypodermis might be of epidermal origin (Fig. 5F). The cells walls are thickened and the tangential wall is covered by a cuticle. While in O. heterophyllum, the cells of the outer epidermis are mostly cubical with occasionally sinuate anticlinal walls, in M. cuneifolia they are cubical in younger stages and become tangentially elongated in mature fruits, where, in longitudinal section, their periclinal walls appear longer than their anticlinal walls (Fig. 6B). The hypodermis was in both species made of cells clearly differentiated from the mesocarp. In M. cuneifolia, it is made of one or two, occasionally three layers of cells. Their cytoplasm appears either very dense and dark or transparent with globular unstained inclusions (Fig. 6A-B). Some cells are transitional between these two types so that my hypothesis is that in the former ones, the same globular inclusions are present in very high concentration. The IKI test gave negative results for starch. In the same way, when observed in polarised light no presence of crystals in the hypodermis was detected. It might be possible that the chemical nature of the cells is lipidic. In O. heterophyllum the

23 number of layers of the hypodermis is variable in different parts of the same fruit (Fig. 7A). The cells are significantly bigger than the cells of the outer epidermis, the shape is more irregular, and the periclinal divisions are more pronounced than in M. cuneifolia. In addition, the same type of cells can be found scattered in the mesophyll as idioblasts (Fig. 7B).

Figure 4 O. heterophyllum. A,C= LS of young fruit showing details of pericarp and seed. B,D= TS of fruit at the same stage of development. Only one of the carpels only is fertile and a single ovule develop into a seed. The chalaza is taking over the protection of the seed at the expense of the single integument. The subepidermis remains one-layered and do not participate in the formation of the endocarp. Oe= Outer epidermis; M=ovarian Mesophyll; Ch=Chalaza; Fu=funicle; Es=Embryo sac; red arrow=hypodermis; white arrow=subepidermis of inner epidermis; asterisk= integument.

When stained with Toluidine blue, their cytoplasm appears dense and light to dark blue which usually indicates that the dye reacted with polyphenolic substances such as lignin and tannins. Similarly to M. cuneifolia, IKI test provided negative results for starch. The presence of lignin or tannins in cells scattered in the mesophyll, would explain why the fruits of O. heterophyllum would break easily during sectioning compared to M. cuneifolia. No such structures as the ones found in the hypodermis of M. cuneifolia were observed. Likewise, these cells might contain mucilage. In both O. heterophyllum and M. cuneifolia the mesocarp is multi-layered and parenchymatous with cells irregular in shape. During development the cells of the mesocarp divide in different planes, develop many intercellular spaces and decrease considerably in size towards the contact zone with the endocarp. Small and inconspicuous vascular bundles developed in this region. Vascular bundle elements include tracheids presenting helical thickening. At the oldest stages of M. cuneifolia observed, the mesocarp is differentiated into three regions: cells in the external region are isodiametric, they tend to elongate radially towards the endocarp in the median region and they become considerably smaller at the contact with the endocarp (Fig. 6A). Such differentiation was not observed in O. heterophyllum. Crystalliferous cells were found dispersed throughout the mesocarp in both species, though in higher concentration in M. cuneifolia, particularly in proximity of the placentation region. The endocarp is derived from the inner epidermis by mean of anticlinal and periclinal divisions of the cells (Fig. 3B, D). In M. cuneifolia, for some time during development the cell walls and cytoplasm are clear when observed at the microscope and are of parenchymatous nature. Later on, the cells begin to differentiate and lignify (Fig. 5D). At the most mature stage observed, they are completely lignified. At this stage they are best defined as sclereids with very wavy outlines (Fig. 6D). As for O. heterophyllum, at the stages available, the endocarp was multi-layered and the cells of the endocarp did not start lignifying yet (Fig. 7). The cells are large in the middle layers and become smaller, tangentially elongated and with visible nucleus in the outer layers. These zones are of high division activity.

Figure 5 M. cuneifolia. LS of young fruits showing pericarp anatomy. A= Carpels equally developed bearing two ovules each with marginal placentation. B= The unequal development of the carpels changes the original orientation of the pericarp and seed. C= Later stage where differentiation takes place in the pericarp layers. D= detail of the persistent one-layered subepidermis and of the lignifying endocarp whose cell walls are subjected to mechanical pressure and become wavy. E= close up of the exocarp showing hypodermis and outer epidermis with anticlinal division. F= Close up of the exocarp showing inclusions in the hypodermal cells and of outer epidermis showing periclinal division. Ov=ovule, Vb=vascular bundle, Oe=Outer epidermis, M=mesocarp, En=endocarp, X=aborted carpel, Black arrow= subepidermis, Red arrow=hypodermis, Black arrow head=anticlinal division, Red arrow=periclinal division. Fu=Funicle, Pl=Placenta, Pd=pedicel, X=aborted carpel, Oe=Outer epidermis, Hy=Hypodermis, Arrow= inclusions, Arrow head=Vascular bundle

Figure 6 M. cuneifolia LS of old stage. A= Well differentiated pericarp and lignified endocarp, with the orthotropous seed enclosed in the vestigial seed coat. B= Close up of the exocarp with details of the hypodermis showing inclusions and of the epidermis tangentially elongated and covered by a cuticle. C= Same details of a slightly younger stage with epidermis not yet subjected to stress forces and hypodermis with same inclusions. D= Detail of the simple vascular bundle and typical crystal found in the mesocarp. E= Detail of the sclereids with wavy walls forming the endocarp. Ex=Exocarp, Me=Mesocarp differentiated into three regions, En=Endocarp, Sc=Seedcoat, Es=Endosperm, Ch=Chalaza, Fu=Funicle, Pl=Placenta, Pd=pedicel, X=aborted carpel, Oe=Outer epidermis, Hy=Hypodermis, Arrow= inclusions, Arrow head=Vascular bundle.

3.2 Seed development

Both M. cuneifolia and O. heterophyllum possess initially two ovules in each of the two carpels. In M. cuneifolia the ovules initiate next to each other but are superposed in mature flowers (Ronse De Craene and Wanntorp, 2008). In both species, since the early stages of fruit development, the two ovules of the aborting carpel and one of the developing carpel degenerate. The ovules of both M. cuneifolia and O. heterophyllum are orthotropous to hemianatropous and crassinucellar with a single integument (Fig. 4, 5A). The placentation is axile and the ovules are attached to the placenta by a very short, almost absent funiculus. In O. heterophyllum the integument is 3-4 layers and non-multiplicative (Fig. 4C). The chalaza is well developed so that the integument initiates half of the length of the ovule. No micropyle was directly observed at the ovule stage. The immature seed is characterised by reabsorption of the nucellus and formation of the endosperm. The differentiation of the single integument into a seed coat was not observed. Instead, the chalaza grows in all directions and builds by intercalary growth a new container for the endosperm and embryo, relegating the integuments at a vestigial state at the now visible micropylar end of the seed which is at the opposite end of the funicle (Fig. 7C). At this stage, the seed is pachychalazal, with a not yet developed embryo and conspicuous endosperm (Fig. 7A). The lack of clear cells in the embryo sac suggest that the endosperm might be of nuclear type in early stages of development. Later in development, the endosperm is cellular with cells that are globular, as large as the ones of the endocarp and develop many intercellular spaces. They divide in all planes but an internal meristematic zone is present at the contact with the embryonic tissue. The mature seed of O. heterophyllum was not available for observation but the other species showed seeds that were exalbuminous (Fig. 9C). The mature embryo consists of a long, thick and curved hypocotyl-radicle axis with a highly folded cotyledons and, as shown by the IKI test, is rich in starch. In these species, a seed coat-like structure was always present between the embryo and the endocarp as a thin inconspicuous layer. Contrary to O. heterophyllum, the integument of M. cuneifolia presents some differentiation during development but the layers tend to become progressively less distinct and are eventually crushed (Fig. 8). A confident classification in testal and tegmic seeds according to Corner (1976) was not possible due to the lack of data. On the other

28 hand, the single integument found in the unitegmic species of Meliosma has been hypothesised to be the inner one. (Ronse De Craene and Wanntorp, 2008).

Figure 7 O. heterophyllum old stages. A= TS of older stage but not mature fruit showing the pericarp differentiation, abundant endosperm and immature embryo. B= LS showing details of mesocarp and not yet lignified endocarp. C= LS close up on micropylar end of the seed bearing a rudimental integument.

If that is true, the seed would be best described as tegmic. The tegmen would be differentiated into an external layer of thin walled, tangentially elongated cells with visible nucleus, a loosen layer of cells below this first layer, a layer of cubic cells of lignified walls, a layer of large, radially elongated cells with thin walls and other two-three layers of loosened, tangentially elongated cells. Although a micropyle was not directly observed, the seed of M. cuneifolia seems to be orthotropous, as in O. heterophyllum, with the chalaza lying on the same line of the funicle and these two region found next to each other (Fig. 6A). The absence of the raphe supports this latter hypothesis. In M. cuneifolia the chalaza does not participate in the formation of the seed cover as it does in O. heterophyllum, but remains located below the seed coat. The cell walls of the placenta thicken and lignify. Cells with inclusions that looks very much like the one found in the exocarp are found scattered in the chalaza, mixed with tracheids and parenchymatous cells. The endosperm is at least in mature stages of the cellular type, made of large cells with thin walls and clear cytoplasm. Little histological differentiation is shown by the embryo whose cotyledons are parenchymatous.

Figure 8 M. cuneifolia close up of the vestigial seed coat. The layers are progressively crushed during development and do not differentiate any mechanical tissue.

Figure 9 A,C,D= O. maguirei. A= Leathery indehiscent mesocarp and lignified endocarp with reticulate surface showing the line of dehiscence. B= O.eterophyllum (from Zúñiga, 2015). C= Cryosection of the embryo showing the long, thick, curved hypocotyl-radicle axis and highly folded cotyledons. D= Endocarp. E= Sabia paniculata. F= Meliosma alba.

4 Discussion

4.1 Diversification within Sabiaceae

The closest affinities Ophiocaryon-Meliosma formerly based exclusively on morphological evidences, has recently been confirmed by molecular data (Zúñiga, 2015). In the latter study, Sabia was shown to be monophyletic and sister to the clade Ophiocaryon-Meliosma. As already mentioned, Ophiocaryon and Meliosma share several floral and fruit related characters. They both have monosymmetric flowers with two of the five petals reduced, three staminodes and two fertile stamens (Ronse De Craene et al., 2015b), drupes that are subglobose and with a median keel running around the endocarp. Sabia in contrast, has actinomorphic flowers with petals of the same size, five fertile stamens and a drupelets laterally compressed. When comparing Sabia and Meliosma, Ronse De Craene et al., (2015b) found that in the bicarpellate gynoecium of Sabia, contrary to Meliosma, the fusion of the two carpels is often incomplete and postgenital, but in both genera, the early development of the ovules is the same. The ovules are laterally initiated at the same level on the margin of each carpel and later become superposed. A difference between the gynoecia of the three genera is that while the surface of the ovary of Sabia is glabrous (with the exception of S. japonica), the ovary of Meliosma bears unicellular trichomes and so does the ovary of Ophiocaryon in its upper portion (Ronse de Craene and Wanntorp, 2008; Thaowetsuwan, 2015; Ronse De Craene et al., 2015b). All genera have ovules that are orthotropous to hemitropous with a very short funicle, Sabia has only one integument which does not cover the nucellus completely, Meliosma has either one or two (with one of the two very reduced) and Ophiocaryon has only one integument covering the nucellus completely (Ronse De Craene and Wanntorp, 2008; Thaowetsuwan 2015; Ronse De Craene et al., 2015b; this study). A particular case concerns M. alba which, in the phylogeny aforementioned, surprisingly appeared to be sister to the rest of the species of Ophiocaryon sampled. Due to the impossibility of finding specimens, the present study could not test whether the fruits and seeds of M. alba show differences with the other species of Meliosma that might well justified its segregation to its own genus as proposed by Zúñiga (2015). However, Van Beusekom (1971) already considered M. alba to be the least derived species and the

32 explosive pollination mechanism described for Meliosma has been questioned in M. alba (Ronse De Craene and Wanntorp, 2008). The drupes of Meliosma are smaller than the fruits of Ophiocaryon, they show more morphological variation and their endocarp presents a funicular canal which is not present in Ophiocaryon Zúñiga (2015). Anatomically, this study has shown that the pericarp and the seed of the species observed of Meliosma and Ophiocaryon are different in many ways and further investigation on more species might find these characteristics to be apomorphies for either the genera. For instance, while the meristematic tissues giving birth to the different pericarp layers were the same in both species, the idioblasts found in the mesocarp of O. heterophyllum, were not found in the one of M. cuneifolia and, the undifferentiated, crushed seed coat of M. cuneifolia was totally absent in O. heterophyllum.

4.2 Evolutionary trends in Sabiaceae

4.2.1 Fruit development and evolution

The morphology of the fruit of Sabiaceae is relatively simple. The fruit derives from a bicarpellate gynoecium in which one of the two carpel is favoured in its growth while the other become extremely reduced. There is certain reciprocity between pericarp development and seed coat structure (Roth, 1977). In Sabiaceae the pericarp becomes hard and takes over the seed protection. Consequently, the seed coat is reduced and partly disintegrated in the species of Meliosma observed and is completely absent in Ophiocaryon. The pericarp presents the typical differentiation of a drupe and is developed into an exocarp, a parenchymatous mesocarp and a sclerenchymatous, occasionally dehiscent, endocarp of protoderm origin. The sclereids of the endocarp of M. cuneifolia have a very characteristic shape with “wavy walls”, similar to the ones found in other species of Ranunculaceae or in Laurus nobilis L. for instance. It is not surprising to find this character in distantly related taxa as it is the result of mechanical constraints. Wavy outlines are a response to opposing forces developing between neighbouring cells during enlargement (Roth, 1977). The infrageneric classification of Meliosma is currently based mostly on endocarp morphology (Van Beusekom, 1971). The last author considered the endocarps of

Meliosma subg. Kingsboroughia sect. Hendersonia to be the ancestral condition in the genus, in being thin, not fully surrounding the seed and presenting neither a canal nor a groove. He proposed an evolution of the endocarp in the genus towards complexity which occurred by thickening of the endocarp itself and progressive enclosure of the vascular bundle. Following this hypothesis, the endocarp in subgen. Kingsboroughia sect. Kingsboroughia, currently including the bitegmic M. alba and M. veitchiorum, represents a step forward the more complex condition. The endocarp is thicker and presents a groove on its ventral side where the vascular bundle runs and, through a pore, connects to the seed. Subsequently, the most derived type of endocarp is found in subg. Meliosma which has the thickest wall and presents a canal (derived from the groove) for the vascular bundle. Zúñiga (2015) assessed the hypothesis and found that sect. Kingsboroughia is not monophyletic. He then proposes that the ventral groove and oblique pore found in M. veitchiorum could be seen as a transitional state between the undeveloped endocarp of M. alba and the highly developed endocarp of subgen. Meliosma. The more derived endocarp of subgen. Meliosma is further differentiated in sect. Lorenzanea and in sect. Meliosma (to whom M. cuneifolia belongs), in the former the canal is longer and almost perpendicular to the length of the endocarp, in the latter is shorter and almost parallel to the length of the endocarp. This is important because of its effect on the overall shape of the endocarp and, likely, on the final orientation of the seed (see section on seed form and integument). Beside the protection role, the pericarp performs other functions, such as nourishing and seed dispersal. Not surprising is the fact that only one of the ovules develop into a seed as drupes are disseminated as a unit propagule, though multi-seeded drupes are found in nature (Roth, 1977). The lack of dehiscence results in a relatively simple pericarp structure and anatomy with no specialisation in dehiscence mechanisms. Information on dispersal mechanism of Sabiaceae was not found in the literature, however the absence of dispersal syndromes such as wings, prickles, hooks, flotation tissue and hairs, tempts one to the exclusion of anemochory, hydrochory and epizoochory1. Nevertheless, plant dispersal mechanisms are the results of the interaction of several factors and the exclusivity of associations between propagule morphology and individual vectors has been disproved (de Casas et al., 2012). Consequently, we can only assume that Sabiaceae fruits, with their thin fleshy mesocarps are eaten and dispersed by animals.

1 Accidental dispersal by animals that transport seeds on the outside of their body rather than inside. (Van der Pijl, 1969).

As for the evolution of a particular dispersal strategy, one needs to take into account that the strategies are the result of a variety of selective forces acting on lineages in the past, some of which acted to increase dispersal rates and distances, while others acted to reduced them (Cousens et al., 2008). A fundamental background that enables one to approach such a topic would include not only a phylogeny of many representative species of families of Proteales and neighbouring families but also data on past distribution, autoecology and synecology of these taxa.

4.2.2 Seed form and integuments

Bitegmy has been considered to be the most primitive condition among angiosperms and unitegmic ovules are thought to be derived from the bitegmic ones (Sporne 1969; Endress, 2011). Families among basal eudicots with both bitegmic and unitegmic species are, among others, Menispermaceae and Ranunculaceae (Corner, 1976). Three processes have been described to be involved in the evolutionary pathway from bitegmy to unitegmy: suppression of one of the two integuments, fusion of the two integuments and integumentary shifting2 (Bouman and Calis, 1977). In basal angiosperms and some derived groups unitegmy has evolved probably mainly by loss of the outer integument, while in derived eudicots mainly by fusion Endress (2011). Ronse De Craene and Wanntorp (2008), observed that in Meliosma veitchiorum Hemsley an outer integument initiated but failed to develop. That would support the idea that the shift to unitegmy in the genus and possibly in the family might have occurred by loss of the outer integument. Some features of the integument, in particular its initiation and mode of growth are considered to be taxonomically significant (Bouman, 1971). This is because similar structure of the mature seed coat might have arisen from different histogenetic process and not be homologous. Consequently, the structures that one wants to compare are not suitable for comparison. In the case of Sabiaceae, a comparative analysis of the complete ovular histogenesis, with particular focus on the integument(s) are even of greater importance as the mature seed coat -that usually provides important information- can be absent (as in O. heterophyllum) or failed to develop any mechanical layer (as in M. cuneifolia).

2 A complicated ontogenetic process involving primordium fusion, a shifting of the i. i. and an arrested growth of the i. i. (Bouman and Calis, 1977).

Endress (2011) underlines how curvature and advent of bitegmy are initially functionally connected as the outer integument is responsible for curvature. One fact supporting this idea is that the outer integument is often thinner in orthotropous ovules than in anatropous ones, or is even absent. In those Proteaceae and Platanaceae species with orthotropous ovules the outer integument is only 2-3 layers, in other anatropous Proteales it is thicker (e.g. Nelumbonaceae A.Rich.). Sabiaceae fits this theory in having hemitropous to orthotropous ovules with a much reduced outer integument or an integument only, the latter being probably the result of the loss of the outer one. The shape of the seed is mostly affected by the ovule orientation but it is also determined by the position of the ovule and the direction in which the ovarian loculus is extended by intercalary growth (Corner, 1976). In Sabiaceae the orthotropous seed is a reflection of the original curvature of the ovule but it is also the result of the modality of growth of the pericarp. The unequal development of the carpels causes the original orientation of the seed to change. If the ovule that develops into the seed, in all the fruits or in some of them is a hemitropous ovule rather than an orthotropous one, the locule shape structure could explain the final orthotropous seed form. As aforementioned, at least in Meliosma, the endocarp shape is currently used for infrageneric classification and it is thought to have evolved towards complexity. Further investigation on the seed type of Meliosma spp. could assess the effect that the endocarp evolution has had on the seed orientation. In Ophiocaryon, beside unitegmy and ortho/hemi-tropy, an additional phenomenon occurred in the evolutionary history of the genus. The single integument of the species observed, lost its function and has become vestigial in the form of a little appendage at the end of the micropylar end of the seed. The process by which this transformation happened involved the chalaza. During seed ontogeny, the chalaza develops by intercalary growth and forms a new container for the seed while pushing the non-multiplicative integument far away from the short funicle. Pachychalazaly occur scattered in several angiosperm groups (Endress, 2011) and is considered to be a polyphyletic advance in seed construction. The basal growth proper of pachychalazal seeds is the antithesis of the acropetal growth proper of dicotyledonous organs and considered primitive (Corner, 1976). The mature fruits of the Ophiocaryon species used for anatomical investigation were not available for direct observation and the seed-coat like structure found in other species is

36 possibly lacking. An image of O. heterophyllum in Zúñiga (2015) shows no such a structure, though it might have been removed by the author. If the seed coat is present, then it might be derived from either the inner layer(s) of the endocarp prior to lignification, or from the outer layer(s) of the seed, that would be the expanded chalaza itself. Otherwise, the other species of Ophiocaryon might not be pachychalazal and their single integument might persist and eventually differentiate in the rudimentary seed coat observed. Similarly, it was not possible to confirm the presence of such a structure in the mature fruits of M. cuneifolia.

4.3 Comparison with closest families

One seeded drupes can be found in Proteaceae in the tribe Persooniinae of the subfamily Persoonioideae (sensu Johnson, 1975). The tribe includes drupes similar to Sabiaceae drupes, with a parenchymatous mesocarp without sclereids and more or less differentiated into three layers, a non-crystalliferous stony endocarp of inner epidermal origin and a vascular system weakly or not developed. Johnson (1975), believed drupaceous fruits in Proteaceae to have repeatedly evolved from follicular types, as a result of a secondary adaptation to endozoic dispersal (bats and birds) in the rainforests. Menispermaceae also produce single-seeded drupes, consisting typically of a coriaceous exocarp, a fleshy or fibrous mesocarp, and a woody endocarp and derived from a biovular gynoecium in which one of the ovule aborts and the other one develops into a seed (Wefferling et al., 2013). If the fruit is a structure particularly subjected to different ecological pressures with repercussions on the seed morphology and anatomy, the embryological characters have been pointed out to be reliable indicators of taxonomic affinities and evolutionary tendencies in flowering plants (Johri et al., 1992). Comparative studies of representative species of families of early diverging eudicots including several anatomical, morphological, embryological characters were considered in this paragraph in an attempt to find linking characters with Sabiaceae and, the results of this search, are summarised in table 1. The gynoecia of the families of the orders Ranunculales and Proteales are mostly superior and, contrary to Sabiaceae, apocarpic with a few or many carpels (Endress and Igersheim, 1999).

In Ranunculaceae syncarpous gynoecia occur, though rarely and withouth a compitum (Endress and Igersheim, 1999). Carpels are reduced to one in some species of Menispermaceae and Ranunculaceae and in most Proteaceae. Sabiaceous characters found in some Ranunculaceae are oxalate druses in the carpel (Igersheim and Endress, 1998; Endress and Igersheim, 1999); the presence of two ovules in each carpel, of which the upper one develops into a seed and the lower one aborts Endress and Igersheim (1999); crassinucellar and unitegmic ovules (Corner, 1976). Anemoneae and Ranunculeae are generally unitegmic, and the genus Delphinium shows transitional forms (Bouman and Calis, 1977). Hemitropous ovules are also common in some species of Ranunculus. As for other families of Ranunculales, Menispermaceae shares following characters with Sabiaceae: oxalate druses and cells with tanniniferous tissue in the ovary wall (Igersheim and Endress, 1998; Endress and Igersheim 1999); two ovules per carpel, of which the upper one develops into a seed and the lower one aborts; crassinucellar and unitegmic ovules. For instance, the genus Stephania (Menispermaceae) is unitegmic but has a rudimentary outer integument (Bouman and Calis, 1977). Hemitropous ovules are also common in Menispermaceae at least at anthesis (Corner, 1976). As for Proteales, comparison is made with Nelumbonaceae, Platanaceae and Proteaceae. Sabiaceae shares the following characters with some members of Nelumbonaceae: oxalate druses in the carpels; crassinucellar ovules; pachychalazaly (Nelumbo) (Corner, 1976; Igersheim and Endress, 1998; Endress and Igersheim, 1999). Regarding Platanaceae Igersheim and Endress (1998) and Endress and Igersheim (1999) could not find the presence of oxalate druses as in the other families. Similarity was found instead in the presence of orthotropous ovules. Proteaceae is perhaps the member of Proteales which shows most affinities with Sabiaceae. Shared characters are: oxalate druses in the carpels; two ovules per carpel, of which the upper one develops into a seed and the lower one aborts (e.g. Macadamia, Banksia) (Endress and Igersheim, 1999); crassinucellar ovules; hemitropy (e.g. Banksia) (Corner, 1976). Orthotropous ovules are also found in most Proteoideae, some Grevilleoideae, Bellendenoideae, Eidotheoideae (Endress and Igersheim, 1999). As aforementioned, unitegmic ovules are found in some Menispermaceae and Ranunculaceae but not in other Proteales beside Sabiaceae (Bouman and Calis, 1977).

Pachychalazal ovules were instead recorded for some Proteaceae (eg. Macadamia) (Corner, 1976; Igersheim and Endress, 1998). Therefore, although unitegmy is not currently described for members of Proteales, others evidences, based on fruit type, carpels and ovules reduction, presence of druses in ovary wall, ovule curvature, nucellus thickness, pachychalazaly, well justify a basal placement in Proteales. Drupaceous fruits might have been derived from follicle types, and unitegmy as seen in Sabiaceae, might share its ancestry with those Proteaceae bearing orthotropous ovules in which the outer integument is only 2-3 layers.

- -

12 /

seeded)

Drupes

shifting

tropous

Compound of Compound

Integumentary Integumentary

Crassinucellate

Polygonium type Polygonium

Menispermaceae

Hemi/Ana/campylo

; Corner (1976); Bouman (1977); Bouman (1976); Corner ;

tropous

pitum)

type

12 /

Berries

shifting

nucellate

Crassi/tenui

(no com Integumentary

Ranunculaceae

Achenes, follicles, follicles, Achenes,

Hemi/ana

Polygonium/allium Polygonium/allium

- - -

+ 2

ov.)

tropous

Achenes

Platanaceae

Hemi/ortho

orthotropous orthotropous

(o.i reduced in reduced (o.i

Crassinucellate

- -

+ + 2

ov.)

seeded)

Drupes

tropous

Follicles,

roteaceae

Hemi/ortho

orthotropous orthotropous

(o.i reduced in reduced (o.i

Crassinucellate

Polygonium type Polygonium

r of selected characters. Data from present study andpresent study Johnson from (1975) characters. Data selected r of

+ + + 1

type

seeded)

ofo.i?

Drupes

Polygonium Polygonium

Suppression

Ophiocaryon

Hemitropous

Crassinucellate

ous ous

+ +

ov.)

type

12 /

seeded)

ofo.i?

Drupes

tropous

Meliosma

Polygonium Polygonium

Suppression

Hemi /ortho Hemi

orthotrop

(o.i reduced in in reduced (o.i

Crassinucellate

ellate

+ + 1

type

seeded, seeded,

Sabia

ofo.i?

rarely 2) rarely

Drupelets

Polygonium Polygonium

Suppression

Hemitropous

Crassinuc

omparison of Sabiaceae with other early diverging Eudicots diverging other numbe on a with early Sabiaceae omparison of

Ovule Ovule

Process Process

Nucellus

Syncarpy

unitegmy

Fruit type Fruit curvature

involved in in involved

Table (1999); (2011) Endress andand (1998); Endress Endress (1980); Igersheim (1992); Johri VandeIgersheim Water Hoot (1991);

Embryo Sac Embryo

Pachychalazy

Integument(s) Superior ovary Superior

+ +

Curved

fferentiated fferentiated

ruminate

Nuclear, oily, oily, Nuclear,

Pachychalazal

and crushed in in crushed and

Amphitropous, Amphitropous,

Undi

Menispermaceae indehiscent fruits indehiscent

Minut

Tegmen Tegmen

and crushed and

Nuclear, oily Nuclear,

Ranunculaceae

undifferentiated undifferentiated

Linear

crushed

Platanaceae

Tegmen thin, Tegmen

Exalbuminous

uminous

fruits

Cellular

Nuclear / Nuclear

cotyledons

Proteaceae

indehiscent indehiscent

Exalb

and crushed in in crushed and

Undifferentiated Undifferentiated

Foliaceous (flat?) (flat?) Foliaceous

root root

Absent

Cellular

Curved, Curved,

Nuclear? Nuclear?

cylindrical cylindrical

cotyledons

Ophiocaryon

highly folded folded highly

axis with flat, flat, with axis

Pachychalazal

Exalbuminous

Orthotropous, Orthotropous,

hypocotyl

slightly slightly

Curved

Cellular

crushed

Nuclear? Nuclear?

Meliosma

Orthotropous

Tegmic? Thin, Thin, Tegmic?

Exalbuminous

differentiated, differentiated,

root root

+ +

Sabia

dottet”

Curved, Curved,

cylindrical cylindrical

cotyledons

“Testa dark “Testa

highly folded folded highly

Helobial type Helobial flat, with axis

Exalbuminous

hypocotyl

of Table Table of

crystals

Embryo

tissue in in tissue

Oxalate

Seed coat Seed

Seed type Seed

Endosperm

Continuation Continuation

Tanniniferous Tanniniferous ovary/pericarp

Conclusions

Since molecular data have placed Sabiaceae among the early diverging eudicots, the family have been morphologically and anatomically investigated in order to find further support for the different topologies hypothesised. In the most recent phylogeny (Sun et al., 2016) the statistical support has increased for Sabiaceae being at the base of Proteales rather than diverging after Ranunculales. Developmental studies on floral morphology of Meliosma and Sabia have found more similarities with Ranunculales (Ronse De Craene and Wanntorp, 2008; Ronse De Craene et al., 2015a,b) rather than Proteales. The present study could not identify clear synapomorphies linking Sabiaceae and Proteales but represents a step towards the understanding of important evolutionary events that occurred in the family and hypothesise two different scenarios that might be further investigated. There is a clear tendency in the family towards unitegmy. The process involved in this evolutionary shift is likely to be the loss of the outer integument. Another tendency concerns the curvature of the ovule which, if the former theory is correct, would represent the mechanical consequence of the loss of the integument itself. Unitegmic ovules are not found in Proteales but they occur in Ranunculales, though more investigation is needed to evaluate whether the process of loss of the integuments has been the same in the Sabiaceae and in Ranunculaceae and Menispermaceae. Interestingly, some species of Proteaceae and Platanaceae do show orthotropous ovules whose outer integument is reduced. This might represent a link between Sabiaceae and Proteales but again, further developmental studies should be carried out on more species of Sabiaceae and direct comparison with those orthotropous Proteales should be considered. A first phylogeny of Sabiaceae (Zúñiga, 2015) has shed light on the relationships within the family and tested Van Beusekom (1971)’s infrageneric classification of Meliosma. This is important because it allows us to map characters on the phylogeny once an adequate amount of data will have been collected.

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Appendix

Additional material showing photographs and LM pictures of fruits and seeds of Meliosma and Ophiocaryon spp.:

PLATE 1: Meliosma cuneifolia at two different stages of development, when the carpels are equally developed and when one of the carpels aborted and only one of the ovule has developed into a seed. Note that M. cuneifolia belongs to subgen. Meliosma sect. Meliosma of Van Beusekom (1971)’s classification. Its endocarp is considered to be the most derived, with a canal almost parallel to the length of the endocarp.

PLATE 2: Seed coat of M. cuneifolia. Earlier stages than the one showed in the results where the seed coat was crushed.

PLATE 3: Calcium oxalate crystals of M. cuneifolia

PLATE 4: Cotyledons of M. cuneifolia

PLATE 5: Photograph of M. dentate and LM close up on the chalaza-funicle region of M. cuneifolia. Both species present the putative most derived type of endocarp with a canal running through it, but in M. dentata (subgen. Meliosma sect. Lorenzanea) the canal is almost perpendicular to the endocarp.

PLATE 6: O. maguirei, mesocarp, endocarp, seed with the inconspicuous seed coat-like structure and embryo.

Plate 1

Plate 2

Plate 3

Plate 4

Plate 5

Plate 6