MSc Summer Research Project

Floral development of (): understanding the origin of monosymmetry and the epicalyx in the flower

Celina Barroca | August 2014

Thesis submitted in partial fulfilment for the MSc in the Biodiversity and of RBGE | University of Edinburgh 2 MSc Biodiversity and Taxonomy of Plants

Front page: Cuphea procumbens SEM picture

RBGE | University of Edinburgh 3 MSc Biodiversity and Taxonomy of Plants

Abstract

The Cuphea (Lythraceae) is highly variable and floral morphology within this genus is highly diverse. It is also one of the most strongly zygomorphic genera within Lythraceae. Another notable character is the presence of an epicalyx.

The origin of epicalyx within the family is unclear and debatable. Accepting Mayr’s (1969) definition, epicalyx is an “emergence of congenitally fused sepals”. The ontogeny of the epicalyx in Cuphea is very different from that observe in other families such as Dipsacaceae, Rosaceae and Malvaceae and has not been the subject of a comprehensive ontogenic study.

This study has investigated the ontogenic development of the expicalyx and also the monosymmetry observe in six of the genus Cuphea.

This was undertaken using light microscopy and scanning electron microscopy.

It revealed that the epicalyx arises very late in the ontogenic sequence and the monosymmetry of the flower is a result of the loss of a stamen and the development of a large nectary on only one side of the base of the ovary.

The position of the nectary causes the formation of a large nectar sac on the adaxial side of the hypanthium resulting in a strongly zygomorphic flower.

RBGE | University of Edinburgh 4 MSc Biodiversity and Taxonomy of Plants

Table of Contents

Abstract...... 3

Table of Contents...... 4

List of Figures...... 6

List of Tables...... 6

Introduction...... 7

Angiosperms Background...... 7 Flower...... 8 Perianth...... 9 Symmetry...... 9 Merism...... 10 Ontogeny...... 11 Epicalyx – The unresolvable structure...... 12 Study Group...... 15 Aims of the study...... 25

Material&Methods...... 26

Plant Material...... 26 Scanning electron microscopy...... 27

Results...... 28

Morphological observations...... 28 Scanning electron microscopy...... 30 Epicalyx and Symmetry...... 30 Stamen, Gynoecium and Nectary...... 32 Lighting Microscopy...... 34 Petals...... 34 Monosymmetry...... 36 Stamens...... 36 RBGE | University of Edinburgh 5 MSc Biodiversity and Taxonomy of Plants

Nectary...... 38 Discussion...... 41

Comparative analysis of different species ...... 41 Epicalyx origin...... 41

Evolution of the flower in Cuphea: monosymmetry, merism...... 42

Conclusions...... 45

Recommendations...... 46

References...... 47

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List of Figures

Figure 1. Phyllogenetic hypothesis of relationships of the families belonging to 16 the order Myrtale………………………………………….……………. Figure 2. Distribution map of Lythraceae………………………………………… 17 Figure 3. Phyllogenetic tree for the genus Cuphea…………………………………... 20 Figure 4a. Cuphea cyanea flower…………………………………...... 21 Figure 4b. Cuphea cyanea inflorescence………………………………………… 21 Figure 5a. Cuphea micropetala flowers…………………………………………… 21 Figure 5b. Cuphea micropetala inflorescence……………………………………... 21 Figure 6a. Cuphea ignea flower…………………………………………………… 22 Figure 6b. Cuphea ignea solitary flower…………………………………………... 22 Figure 7a. Cuphea caeciliae flower………………………………………………... 22 Figure 7b. Cuphea caeciliae inflorescence………………………………………… 22 Figure 8a. Cuphea hyssopifolia flowers…………………………………………… 23 Figure 8b. Cuphea hyssopifolia inflorescence……………………………………... 23 Figure 9a. Cuphea procumbens flowers…………………………………………… 23 Figure 9b. Cuphea procumbens inflorescence……………………………………... 23 Figure 10. C. caeciliae SEM figure plate...... 31 Figure 11. C. caeciliae SEM figure plate...... 33 Figure 12. C. caeciliae and C. procumbens SEM figure plate…………………… 35 Figure 13. Stamens of fixed dissected half mature flowers plate………………….. 37 Figure 14. Nectary of fixed dissected half mature flowers plate…………………... 39 Figure 15. Floral diagram of Cuphea micropetala...... 40

List of Tables

Table 1. material collection details for each species…………………………. 28

RBGE | University of Edinburgh 7 MSc Biodiversity and Taxonomy of Plants

Introduction

Angiosperms Background

With at least 260,000 living species classified in 59 orders and 413 families (APG III, 2009), the Angiosperms, or flowering plants, are one of the major groups of land plants and the most diverse extant group, comprising around 90% of existent plant biodiversity. However, this diversity in not equally distributed and around 75% of all species belong to the eudicot clade (Soltis et al., 2005, Judd et al., 2007). Angiosperms are far more diverse in vegetative form and in the structure of their reproductive organs than any other group of land plants (Friis et al. 2011).

According to the fossil record based on distinctive angiosperms pollen grain fossils have been dated from early Cretaceous, around 135 million years ago, time of angiosperms origin (Friis et al. 1987, 2011, Judd et al. 2007). Therefore, angiosperms diversified during the Cretaceous period. Many authors suggest different hypothesis and fossil record evidence to date the divergence of angiosperms, evolution and later their lineages e.g according to Friis et al. (2005) the estimated timing of major diversification events in angiosperms range from around 175 million years ago – during Jurassic to around 60 million years ago – at the end of the Cretaceous.

What is certain is, by the end of the Cretaceous, angiosperms have diversified enormously and become widespread, becoming the dominant terrestrial plants on the planet (Magallón and Sanderson, 2001).

Angiosperms have a few synapomorphies (i.e. shared derived characters) that unify and distinguish them from the other groups of plants. These features include:

1) ovules that are enclosed within a carpel (i.e. a structure that consists of an ovary, which encloses the ovules, the style, a stalk portion as a slender region specialized for pollen tube growth and the stigma, a structure which receives the pollen and where pollen germination takes place; 2) double fertilization, which leads to the formation of an endosperm (a nutritive tissue within the seed that surrounds and feeds the developing plant embryo); RBGE | University of Edinburgh 8 MSc Biodiversity and Taxonomy of Plants

3) stamens with two pairs of microsporangia (i.e. pollen sacs); 4) features of female and male gametophyte reduced in size structure and development; 5) phloem tissue composed of sieve tubes accompanied by one or more companion cells (see Doyle and Donoghue, 1986; P. Soltis and D. Soltis, 2004; Judd et al. 2007).

Strong evidence for the monophyly of angiosperms comes from shared derived morphological characters mentioned above but also from molecular studies, contradicting the hypotheses of polyphyletic origins of extant angiosperms.

Flower In general terms, angiosperms are inevitably linked by the structure defined as a flower, although we cannot discard the gymnosperms. Likewise, the definition of a flower changed so many times over time and in the past, the concept of a primitive angiosperm flower could be broadly divided into two main contrasting suites of ideas: the Euanthial Theory and the Pseudanthial Teory, interpreting it as a simple, Cycadales’ strobilus looking-like structure or a compound structure hypothetically homologous to conifer’s cone, respectively. Thus, an inclusive interpretation of the origin and evolution of angiosperm flowers still needs to be defined (Ronse De Craene, L. 2010; Friis et al. 2011).

As mentioned above, gymnosperms also have flowers - reproductive organs homologous with those found in angiosperms but the main difference is that angiosperms flowers generally also have a perianth where in gymnosperms flowers do not. The reproductive organs although highly reduced in angiosperms becoming an evolutionary character crucial in adaptations and has clearly contributed to radiation of angiosperms and flower diversification (Soltis et al, 2005).

For this reason, an understanding of the detailed genesis and functioning of the plant, specifically of the flower, is very important. A comprehensive definition of the flower, according to Weberling (1989), must be:

“a section of a shoot, or a branch resembling a short shoot, which bears leaf organs which serve for sexual reproduction and which are transformed accordingly”.

Therefore, the definition of flower in this work is based on Weberling’s definition and also on the typical angiosperm architecture (archetype), a determinate structure, arranged in whorls RBGE | University of Edinburgh 9 MSc Biodiversity and Taxonomy of Plants or spiral, with a defined number of organs such as sepals, petals (or tepals) – the organs of the perianth, often distinguished as calyx and corolla; stamens and carpels, not always differentiated into outer and inner whorls, but often.

Perianth

The perianth is considered an essential character of the angiosperms flower and it can exists as a simple form (perigon in Monocots with no clearly differentiated whorls) or double form differentiated into calyx which as a protective purpose – sepals, and a corolla which is linked at adaptations for attraction of pollinators, after its size and colour variations – petals. A double perianth is found when there is a distinction between two series or whorls, sepals and petals, although the differentiation between both whorls can be complex due to a) there are cases with more than two whorls of sepals and petals and so tricky to define each whorl, also b) the lobes of each whorl can be joined together laterally (e.g. sepals joined into gamosepalous calyx) or c) organs of different whorls can be united (e.g. stamens joined to the corolla forming a stamen petal tube or epipetalous stamens) (Weberling, 1989; Ronse De Craene, 2010).

Symmetry

Floral symmetry is a structural factor that influences the configuration of a flower and is the result of initiation of flowers organs and their consequent growth and differentiation (Ronse De Craene, 2010).

Tucker (1999) defines it as:

“Floral symmetry is the product of organogeny plus subsequent organ development.”

Flower parts are arranged in different planes and so, when bisected through the centre (floral axis), the flower can have one, two or more symmetric halves, or even have no plane of symmetry.

Resulting from equal, regular growth of organs within whorls, this flower originates two or more symmetrical planes, called radial symmetry, polysymmetry or actninomorphic and pollination can occur from all directions; when organs evolve unequally within a whorl, it RBGE | University of Edinburgh 10 MSc Biodiversity and Taxonomy of Plants becomes differentiated between sides and one side can develop more than the other, being possible to divide it into symmetrical halves on only one plane, defined bilateral symmetry, monosymmetry or zygomorphic, creating a quite common pattern of development among angiosperms, targeting a specific access for pollinators (Zygomorphy can result as it was just described or can be result after a differentiation in size of certain organs in a flower with actinomorphic ontogeny (Tucker, 1999). As just mentioned, organs can develop irregularly and two sides develop differently resulting in two lines of symmetry, called dissymmetry. This pattern is rather rare and found in Papaveraceae or Brasicaceae; (Ronse De Craene, 2010; Judd et al., 2007); last but not least, some flowers have no symmetry plane and are considered asymmetrical. These are the main floral symmetry definitions and for determining it, it is treated the position of the more conspicuous structures such as calyx, corolla and androecium.

Merism

Changes in organ number and position in the different whorls at organ initiation, differential enlargement between floral organs and late differentiation of structures or specific tissues (e.g. the suppression or loss of an organ or the development of a nectary) may affect symmetry during floral development and lead it to zygomorphy (Tucker, 1999).

Zygomorphic symmetry is considered to be a derive condition in the evolution of flowers and actinomorphic symmetry the most primitive. This implies that ontogeny zygomorphy can be expressed at organ initiation or by differential growth (Tucker, 1999).

Payer (1857) mentioned that a lot of zygomorphic flowers have regular initiation and become irregular at later stages. In some taxa, organ initiation is radial and in maturity the flowers are zygomorphic (e.g. Bignoniaceae).

Flower structure and symmetry are directly linked to adaptation to pollination.

Ronse De Craene (2010) subdivides the monosymmetric flowers (after Endress, 1994) into two kinds: a) flag – flowers, which are sternotribic; that means that pollination occurs by the underside of the pollinator throught the stamens that are exposed and curved up; b) lip – flowers, which are nototribic; these flowers have a lip as a landing platform for the pollinator RBGE | University of Edinburgh 11 MSc Biodiversity and Taxonomy of Plants while stamens are usually hidden by the fused upper petals allowing pollination by the back of the pollinator.

A way to attract pollinators is the development/presence of a nectary (i.e. glandular tissue that secretes sugar-rich fluids); it can arise anywhere in the flower and function as a major attractant and a reward (Ronse De Craene, 2010). Nectary evolution is of particular interest since nectar is a very common pollinator reward in extant angiosperms.

Ontogeny

Furthermore, this fusion between members of the same whorl or different whorls may happen from their moment of origin onwards (i.e. congenital fusion)1 or they can develop together later on (i.e. postgenital fusion).

This process of initial development of flowers or posterior fusion knowledge is important not only for a new approach to morphological problems but also for the determination of systematic relationships and its consequent taxonomy.

In other words, flower ontogeny (i.e. the course of initiation and development of an organism and organ till maturity) (Ronse De Craene, 2010), is of extreme importance from an evolutionary point of view because it represents a different set of characters which leads to distinct new information that can be tested against existing phylogenies. Flower ontogeny allows us to reveal information impossible to access without the ontogenic study in order to understand and exceed the limitations of certain species that may be used in different levels of botany.

In this study, I will be looking at the ontogeny of the epicalyx, its origin and structure and also at the origin of monosymmetry within the flower of Cuphea.

1Congenital fusion: fusion of structures from the onset of initiation (zonal growth or ring primordia) (Ronse De Craene, 2010).

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Epicalyx – The unresolvable structure

The epicalyx is a structure of which its origin and definition was study by several authors (Lamarck, 1795; Ventenat, 1795; De Candolle, 1813; Payer, 1857; Köhne, 1873; Eichler, 1878; Troll, 1949; Cadet, 1954; Mayr, 1969; Sattler, 1973) and remains difficult and inconsistent, despite having been the subject of study since 1795. It can be found under other names such as a false calyx, calycle, calyculus, in French “calicule” (De Candolle, 1819), in german “Außenkelch“, etc.

As found in botanical or book glossaries, epicalyx is generally defined as:

“Involucre which contains only one flower, and adheres by its base with the true calyx.”

(De Candolle, 1819)

“A whorl of sepal-like appendages resembling the calyx…”

(Heywood et al., 2007)

“A group or whorl of bracts below the flower that resembles an extra calyx (e.g. in Hibiscus)”

(Beentje, 2010)

“A whorl of bracts or structures of unknown origin inserted at the base of the calyx” (Ronse De Craene, 2010).

Over time different authors have come with different explanations and concepts of the origin of the epicalyx.

Lamarck (1795) defined the differentiation between a “proper” and a “common” calyx: proper calyx can be simple or double; common calyx can be simple, embricate (not relevant) or caliculé. Following the same author, a proper calyx is the one that contains only a single flower and as it was mentioned before, it can be simple or doubled. It is a proper simple calyx RBGE | University of Edinburgh 13 MSc Biodiversity and Taxonomy of Plants when it’s composed of only a single envelope, which is sometimes, or naked, or covered with hairs or spines or even with scales at the base (e.g. Papaveraceae, Primulaceae (Coris) and Dipsacaceae); in contrast, a proper double calyx is composed of two or more remarkable envelopes, all very distinguishable from the corolla (e.g. Malvaceae (Malva and Hibiscus) and Dipsacaceae (Dianthus). Unlike the proper calyx, a common calyx is a structure that contains several flowers arranged in the same receptacle and may still have their own true calyx, giving the example of Compositae; to be a common simple calyx needs to be composed of only one piece as in Asteraceae (Tagetes and Othonna) or composed only of a single row of scales, which overlap each other item (e.g. Tragopogon, Asteraceae); a common caliculé calyx is simple but its outer base is topped with small scales which forms almost a second calyx shorter than the other at least in half.

Lamarck (1795) and Ventenat (1795), they use the expression caliculé calyx to denote a “common” calyx as in Asteraceae (now referred to as an involucre) and Lamarck uses the term double calyx for a “proper” calyx as it happens in Malvaceae (now considered to be as an epicalyx);

With Brisseau-Mirbel (1815, after Cadet, 1954) might be the first time that the epicalyx term actually is define as an outside calyx in a double cup (two calyxes), for both proper and common like in Malvaceae and Dipsacaceae;

For De Candolle (1813), the epicalyx could be a) a very small calyx or; b) a calyx accessory placed outside the true calyx (e.g. Malvaceae) or; c) to express a small row of bracteoles placed at the base of the involucre (e.g. Compositae). Later on he seems to use the term “caliculé” just for the involucres, in his words “when the involucre is composed by bracts in two rows, and the exterior is substantially smaller, we say that the involucre is “caliculé”, or equipped at its base with a kind of small calyx”;

De Saint-Hillaire (1841) defines epicalyx as a set of bracts but he refuses to name the epicalyx whose parts are similar to stipules of the sepals like in Potentilla (Rosaceae);

On the other hand, Payer (1857) and Sachs (1874) suggested that the epicalyx become a sui generis organ exclusive of a flower, although with a variable morphologic value – set of bracts (e.g. Dianthus, Caryophyllaceae and Malvaceae) or stipular appendages of sepals (e.g. Potentilla). RBGE | University of Edinburgh 14 MSc Biodiversity and Taxonomy of Plants

For Van Tieghem (1884) the epicalyx is not more than a set of stipular appendages of the calyx like in Potentilla and he refuses to use the term of epicalyx to unifloral involucre as in Malvaceae, going against De Saint-Hillaire.

The interpretation of certain types of epicalyces, as in Rosaceae, accredited to Roeper (1826, after Cadet, 1954) was applied by Köhne (1873) and he seems to be the first to apply the interpretation of epicalyx to Lythraceae. It is described that certain epicalyces are the result of the fusion of contiguous stipules pairs of two neighbours’ sepals. Moreover, this interpretation was also adopted by Eichler (1878), among other authors.

Troll (1949), did a morphological and ontogenic study on Campanulaceae and he reported the formation of commissural appendages of the sepals in function of the valvate aestivation or “préfloration” associated to a basal growth in between the sepals (e.g. also in Nemophila, Boraginaceae). In this case, he used the same interpretation in Lythraceae, supporting it with the same type of aestivation (valvate) showing to be agreed with Köhne but contradicted Payer.

Mayr (1969) interprets the epicalyx as emergences of the congenital fused sepals.

Within Lythraceae, Cadet (1954) classified the various interpretations of epicalyx in three different groups according to how it is considered the epicalyx parts: its members are equivalent to sepals; they are part of sepals, e.g. like particular appendages of sepals, such as stipules; or they are special growths at the top of the floral tube whose homology is not determined.

As a result of anatomical studies, Cadet (1954) determined that the epicalyx members (calicules) could not represent, both anatomical and morphological point of view, stipular outgrowths of sepals, simple or welded in pairs, either as non stipular outgrowths, yet they are homologous to bracts.

Pluys (2002) mentioned that the structures of Rosaceae, Dipsacaceae and Malvaceae can clearly be called epicalyces, however the origin (especially in Rosaceae) is unclear and the three types are clearly not completely homologous; if an epicalyx is form from emergences, like in Lythraceae, these could be considered a commissural epicalx. Based on Mayr’s (1969) description, Pluys (2002) consider that the main characteristics of the commissural epicalyx RBGE | University of Edinburgh 15 MSc Biodiversity and Taxonomy of Plants are the large variation in occurrence and shape, and the fact that it hardly resembles a leaf although without clear homology.

Study group

The group of plants in question in this study belong to the core , which comprises the vast majority of eudicot species and with seven major clades, one of which is .

The rosids comprise 140 families and is close to a one-third of all angiosperms species, although have not been identified clear synapomorphies for this clade, relying in morphological and anatomical characters like nuclear endosperm development, reticulate pollen exine, two or more whorl of stamens, etc (Soltis & Soltis, 2004).

According to molecular analyses, two main subclades of rosids were identified – eurosids I – the fabids and eurosids II – the malvids. In spite of this, some orders could not be placed in either subclades as is the case with .

The order Myrtales Reichenbach consists of approximately 6% of the diversity in core eudicots (Magallón et al, 1999; Kubitzki 2007). This order has not had massive changes in its phylogenetic composition and remains relatively congruent with more recent concepts. De Candolle (1828, after Kubitzki, 2007) circumscribed the major myrtalean families into Myrtales as we still know nowadays. Thus, and after extensive studies of some authors mentioned below, the relationships within the order in the present are represented in Fig.1 below. RBGE | University of Edinburgh 16 MSc Biodiversity and Taxonomy of Plants

Fig. 1. Phyllogenetic hypothesis of relationships of the families belonging to the order Myrtales, studied by by Conti et al. (1996, 1998, 1999, 2002), Sytsma et al. (1998, esp. 2004), Clausing and Renner (2001: Melastomataceae), Schönenberger and Conti (2001, 2003: esp. Penaeaceae area, etc.) and Wilson et al. (2005: Myrtaceae s.l.) (Stevens, 2001 onwards)

The order is characterised by their often flaky bark; opposite leaves with undivided laminas; small or rudimentary stipules; short to elongated hypanthia (usually nectariferous); sepals often valvate; clawed petals or really narrowed at the base; stamens incurved in bud; etc. From a typological point of view, the inflorescences are monotelic thyrsopaniculate, claimed to be basic in the order (Kubitziki, 2007). The basic chromosome number may be x = 12. In particular, Myrtales has been defined by the combined occurrence of vestured pits and bicollateral vascular bundles in the primary xylem and its current distribution belongs to tropical, subtropical and warm-temperate regions of the Southern Hemisphere. (Graham et al. 1993; Conti et al. 1997; Stevens, 2001 onwards). RBGE | University of Edinburgh 17 MSc Biodiversity and Taxonomy of Plants

One of the families within the order is Lythraceae (Jaume Saint-Hilaire, Expos. Fam. Nat. 2:175 (1805), nom. cons.) (Brummit, 1992).

The plants of this family are found throughout the tropics, extending into temperate regions (not generally present in African and Arabian deserts and high latitudes) and geographically divided between the Old World (18 genera) and the New World (13 genera), with higher level of representatives in tropical America and Africa (Heywood et al, 2007, Graham et al. 2005).

Fig. 2. Distribution map of Lythraceae (Stevens, (2001 onwards

Lythraceae sensu lato is a highly diverse family comprising 31 genera distributed worldwide and around 600 species within four subfamilies: Punicoideae (Punica or pomegranate); Duabangoideae (Duabanga); Sonneratioideae (Sonneratia); and Lythroideae (28 genera and Lythraceae s.s) (Graham et al. 1993).

Moreover, the type of habit and habitats include tall to small trees, or perennial to annual herbs adapted to a broad variety of vegetation types from mangrove swamps, rainforests to fresh-water marshes. They are characterised with leaves opposite, often decussate, simple, usually entire; stipules, when present, small and arising in the leaf axils. Inflorescences are terminal or axillary, in racemes, panicles, cymes or rarely solitary. The flowers are monoecious, radially symmetrical to strongly zygomorphic, hermaphrodite with development of floral tube that diverges from widely campanulate to tubular hypanthia. An epicalyx is sometimes present, alternating with the sepals. The sepals are 4 – 6 (-16), joined RBGE | University of Edinburgh 18 MSc Biodiversity and Taxonomy of Plants at the base, valvate, often with distinct external ridges. The petals are 4 – 6 (-16), creased in bud, often bright coloured, or absent. The stamens are usually twice as many as sepals (diplostemonous androecium), attached to the calyx tube in two whorls, free, usually alternating short and long. The ovary is usually superior, situated in a free hypanthium, 2 – 4 (- many) fused carpels, each forming a locule, with one to many ovules per locule, with a single and dry style, placentation axile. The fruit can be a dry dehiscent capsule to a fleshy- seeded capsule or berry. Distyly and Tristyly are present. This heterostyly occurs in six genera and distinguishes the family (Graham et al., 1993, Heywood et al., 2007).

Lythraceae has been placed in Myrtales according to the main traditional systems of classification and APG III. The genera within the family are mainly well defined although there is still some discussion going on (Heywood et al., 2007).

Köhne (1903) proposed that in Lythraceae s.s should be recognised two tribes based on ovarian septa structure but happens that fossil evidence and some additional studies show that it was incorrectly described (Graham et al., 1993).

According to Mayr (1969) in his study of the order, Lythraceae are characterised by a perianth tube between the whorls of calyx and corolla and the stamen whorls; he also mentions that the petals develop with a great temporal difference, shortly before anthesis and the androecium of all taxa is diplostemonous and in any case of obdiplostemony, its appearance depended on primary differences of size and secondary changes in growth. In addition, the retarded genesis of petals and the obdiplostemonous appearance of the androecium is seen as a result of a successively reduced segment of petal. The structure in study – epicalyx – is interpretated by Mayr (1969) within many species of Lythraceae as an emergence of the congenitally fused sepals (compared with commissural stigma).

One of the genera in the family Lythraceae and the one in study is Cuphea P.Browne (Civ. Nat. Hist. Jamaica: 216 (1756)) (Kubitzki, 2007)

Cuphea (c.260 spp) is certainly the largest genus in Lythraceae, restricted and endemic to the New World; classified in two subgenera and thirteen sections. The genus is described as herbaceous perennial and small shrubs and differentiated from other herbaceous genus of the RBGE | University of Edinburgh 19 MSc Biodiversity and Taxonomy of Plants family by the zygomorphic ribbed floral tube with six deltate calyx lobes and a unique seed dispersal mechanism (in comparison to other Lythraceae genera, Cuphea seeds are exposed for dispersal on a placenta that becomes exserted throught matching longitudinal slits in the adaxial (dorsal) wall of the capsule and floral tube). Synapomorphies of the genus are 11 stamens; unilateral free-standing nectariferous organ, at the base of the ovary; septa reduced to thin threads; oblate pollen and seed oils (Graham et al. 2006, Melazzo and Oliveira, 2012).

Cuphea is described with opposite, simple leaves; bracts present; inflorescence in racemes or thyrses, with one flower always interpetiolar at a node, the others on axillary branchlets; floral tube cylindrical of varying length, bilateral, 12-nerved, green, red or purple, zygomorphic and often spurred at the base; sepals very short with appendages present – the presume epicalyx; petals 6(- 0); stamens 6(-11); ovary subtended by a free-standing unilateral nectar; 2 locular, 1 reduced, septa filiform; capsule dry, thin-walled, dehiscing by a dorsal longitudinal slit; chromosome number x = 8 (Tobe et al. 1998; Kubitzki, 2007; Graham, 2009)

An epicalyx of “appendages” positioned on the floral tube at the sinuses of the sepals (margins of the calyx lobes are congenitally fused and enlarge) is present in many species (Mayr,, 1969; Tobe et al. 1998).

Cuphea has multicellular hairs – Trichomes - secreting resinous exsudates; has also others unicellular types including malpigiaceous cystolitic hairs and globose glands with elongated necks or with spinulose surfaces (Tobe et al. 1998).

Geographically, its distribution can be divided between two major centres as in North America, western and southern of Mexico; and in South America, eastern Brazil. Most species occupy mesophytic to marsh habitats but can also be found, especially narrowly distributes endemics, in white sand savannas, rocky limestones outcrops and swamplands.

Diversity is registered in both distribution centres, being vegetative morphology in South America and flower morphology has widely diversified in Mexico (Graham et al. 2006).

Cuphea is a monophyletic genus closely related with other genera such as Adenaria, Pehria, Koehneria, Woodfordia and Pleurophora, but the sister group remain unclear (Graham et al. 2006)

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Fig.3. Phylogenetic tree for the genus Cuphea (strict consensus of 220 parsimony trees based on a heuristic search of the ITS dataset) Species endemic to North America are in bold typeface and South American species are in plain typeface. Numbers above the branches are jackknife values ≥ 50% (Graham et al. 2006).

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The species of Cuphea studied are Cuphea cyanea DC; Cuphea hyssopifolia Kunth; Cuphea caeciliae Koehne; Cuphea ignea A.DC.; Cuphea micropetala Kunth; and Cuphea procumbens Ortega.

Fig. 4a. Cuphea cyanea flower (floral tube Fig. 4b. Cuphea cyanea inflorescence length = ± 2.2 cm). (thyrse).

Fig. 5a. Cuphea micropetala flowers (floral Fig. 5b. Cuphea micropetala inflorescence tube length = ± 2.5 cm). (raceme). RBGE | University of Edinburgh 22 MSc Biodiversity and Taxonomy of Plants

Fig. 6a. Cuphea ignea flower (floral tube length Fig. 6b. Cuphea ignea solitary flower. = ± 2.5 cm).

Fig. 7a. Cuphea caeciliae flower (floral tube Fig. 7b. Cuphea caeciliae inflorescence length = ± 2.5 cm). (thyrse - raceme with uniparous cymes). RBGE | University of Edinburgh 23 MSc Biodiversity and Taxonomy of Plants

Fig. 8a. Cuphea hyssopifolia flowers (floral Fig. 8b. Cuphea hyssopifolia inflorescence tube length = ± 0,5 – 0,6 cm). (thyrse).

Fig. 9a. Cuphea procumbens flowers (floral Fig. 9b. Cuphea procumbens inflorescence tube length = ± 2.5 – 3 cm). (thyrse).

All the Cuphea species illustrated show the position of the large nectar sac positioned on the adaxial side of the flower and also the stamens and style on the abaxial side of the flower giving to these flowers a distinct zygomorphic monosymmetry. RBGE | University of Edinburgh 24 MSc Biodiversity and Taxonomy of Plants

Several species of Cuphea have use and economic importance and are well-known Latin American folk remedies for syphilis, fevers, diuretics or laxatives. Cuphiin, a macrocyclic tannin isolated from C. hyssopifolia, shows promising anti-tumor activity; they are very popular as ornamental.

RBGE | University of Edinburgh 25 MSc Biodiversity and Taxonomy of Plants

Aims

Floral ontogeny is important for a better understanding of floral evolution within the genera, family and order – in this work, morphological analysis carried out on light photography, light microscopy (LM) and scanning electron microscopy (SEM) techniques of different ontogeny stages were used to identify the origin and evolution of floral organs.

The aim of this study was to observe and analyse the epicalyx and the evolution of symmetry, with the main approach to presenting a complete ontogenetic sequence of different organs of six different species of Cuphea, as a member of Lythraceae family and to compare it with other already examples of studied families, and so understand the evolution of the epicalyx and monosymmetry in the flower which were the main focus of observations, and answer specific questions.

Floral ontogeny represents a separate set of characters that can be tested against existing phylogenies; it reveals hidden evidence that exceeds species limitations and can be used at different levels.

RBGE | University of Edinburgh 26 MSc Biodiversity and Taxonomy of Plants

Material&Methods

Morphological study

Plant Material

Fresh plant material was obtained from the collection in the greenhouses of the Royal Botanic Garden of Edinburgh (Table 1). Fresh inflorescences with early buds to mature flowers were fixed in FAA (70% Ethanol, EtOH; 5% Formaldehyde, CH2O; 5% Acetic acid, CH3COOH).

Table 1. Plant material collection details for each species.

RBGE Acc. Collected Collected Species Living plants of this accession Number in by

Compton, Mexico : J.; D'Arcy, Cuphea cyanea DC. 19933097 19933097D: G04 Arid Lands Nuevo J. & Rix, León E.M. 1321

19671788B: G21 Temperate Cuphea hyssopifolia 19671788 Lands Kunth 19671789A: G16 Temperate 19671789 Lands Breedlove, D.E. & Cuphea caeciliae 19822285B: G17 Temperate Mexico : Bartholom 19822285 Koehne Lands Chiapas ew, B. 55679

19696576E: G35 Orchid & Cuphea ignea A.DC. 19696576 Cycads Dunbar, Rebecca; Harley, Cuphea micropetala 19960237A: G71 Temperate Struan; 19960237 Lesotho Kunth Collection Hirst, M. & Webster, D. 207

Cuphea procumbens 20090221 20090221A: G04 Arid Lands Ortega

RBGE | University of Edinburgh 27 MSc Biodiversity and Taxonomy of Plants

The fixed material (i.e. young flower buds and inflorescences) was dehydrated in 70% EtOH, observed and dissected under a dissecting Light Microscope (ZEISS Stemi SV6 Dissecting Microscope) ready prepared for the next method.

Scanning Electron Microscopy

Following the Critical Point Drying Method using FAA protocol the material selected previously was rinsed in 70% EtOH, transferred into baskets and dehydrated through an

EtOH/distilled H2O series:

70% ...... 15min

95% ...... 10min

100% ...... 5min

Once the specimens were acclimatized for a stronger solution, they were transferred into Acetone:

100% Acetone.....5min x 2

This procedure took place on a ventilated bench.

At this point, samples were ready for critical point dried with liquid CO2 using an Emitech K850 Critical Point Drier. When finished, specimens should be mounted into SEM carbon- coated sticking tabs in SEM stubs and sputter coated with platinum using an Emitech K575X Sputter Coater machine.

Next, sequences of different stages of flower development were observed and photographed using a LEO Supra 55VP Scanning Electron Microscope.

SEM images were processed using Adobe Photoshop CS6 and plates designed with Microsoft Office Publisher 2007.

Living collection pictures were taken with a D3200 Nikon HDSLR Camera and processed with Microsoft Office Picture Manager 2007.

Fixed and half dissected flowers were photographed using a ZEISS Stemi SV6 Dissecting Microscope and a Zeiss Axiocam MRc 5. RBGE | University of Edinburgh 28 MSc Biodiversity and Taxonomy of Plants

Results

In this study, flowers and inflorescences of six Cuphea species were studied.

The species in question are:

 Cuphea cyanea DC;  Cuphea hyssopifolia Kunth;  Cuphea caeciliae Koehne;  Cuphea ignea A.DC.;  Cuphea micropetala Kunth;  Cuphea procumbens Ortega..

Morphological observations

The morphology of this genus is highly diverse but all shared the following characters:

 Tubular hypanthium;  Six calyx lobes – sepals;  “epicalyx” emergences;  Superior ovary;  Presence of nectary (although more reduced in some species than in others) in the lower part of the ovary;  Opposite (decussate), entire and simple leaves;  Stamens insertion on the floral tube;  Presence of trichomes;  Monosymmetry, although some are more strongly zygomorphy than others.

RBGE | University of Edinburgh 29 MSc Biodiversity and Taxonomy of Plants

Some main morphological differences are:

 Calyx lobes size and shape;  Corolla lobes – petals size and shape when present, frequently absent;  Level insertion of stamens, length and shape;  Length of the style;  Colour and shape of the hypanthium;

All species have inflorescences in thyrses, racemes with one flower always interpetiolar at a node and the others on axillary branchlets (cymes), or solitary (C. ignea); small to large corolla - petals (C. procumbens, C hyssopifolia, C. cyanea).

RBGE | University of Edinburgh 30 MSc Biodiversity and Taxonomy of Plants

SEM - Epicalyx and Symmetry

Plate 1

Cuphea caeciliae

Development of the calyx and epicalyx

A. Top view of a three young flower buds plus its bracts; B. Young bud of the first calyx primordia, calyx lobes – sepals primordia (*) – regular whorl initiation; C. Slightly older bud with calyx primordia clearly conspicuous; D. Later stage with sepals development; the bud is concave by the beginning of the hypanthium (can I call it hypth. already?) growth; E. All sepals are constructed; disymmetry clearly visible indicated by arrows; F. Alternating with the sepals appears the calyx appendages – the theoretical epicalyx (Ec); at the top pf the sepals appears the first hairs; G. Hypanthium with well visible calyx lobes alternating with its appendages (Ec); H. Inflorescence.

Key for images: Sp = Sepals; Ec = Epicalyx

As it can be seen in plate 1 (Fig10, A – H), two bracts arise to protect the flower bud (Fig. X, A) and the calyx initiation appears simultaneous with six sepals (Fig. X, B – C). The hypanthium starts to expand with the six calyx lobes developing and bending towards the centre as they form (Fig.10, D – F), forming a valvate aestivation. Unfortunately, there is no data available for the earlier stages of initiation. The symmetry of the flower bud starts to change from radially symmetric to a disymmetric calyx (Fig. X, E). Emergences between the calyx lobes appear at the top of the hypanthial tube(Fig.10, F – G) forming the theoretical epicalyx. The inflorescence type found is a thyrse (Fig. 10, H).

RBGE | University of Edinburgh 31 MSc Biodiversity and Taxonomy of Plants

Plate 1

Fig.10. C. caeciliae SEM figure pla RBGE | University of Edinburgh - 32 - MSc Biodiversity and Taxonomy of Plants

SEM – Stamen, Gynoecium and Nectary

Plate 2

Cuphea caeciliae: Development of the stamen whorls, gynoecium and nectary

A. Young flower bud with the calyx removed to show the position of stamens and gynoecium initial formation; stamens arising in two whorls; B. Calyx partially removed to show the position of the stamens whorls relatively to the sepal lobes; arrow is indicating the lack of the stamen of the second whorl; C. Slightly older bud with stamens and carpel growth; differentiation of the wall of the gynoecium which will form the nectary tissue; D. Top view of a intermediate stage development of stamens and gynoecium showing the big gap of the absence of a stamen;

E – F. Stamens well developed, already forming the anthers; nectary position formation clearly visible.

Key for images:

* = first whorl of stamen primordia

** = second whorl of stamen primordia

G = Gynoecium and gynoecium primordia

N = Nectary primordia

Arrow indicates the lack of a stamen primordia from the second whorl

According to plate 2 (Fig. 11, A – H), the two stamen whorls have initiated as well as the gynoecium (Fig.11, A); because the earlier stages of initiation were not observed, the apparent centrifugal initiation cannot be relied upon. The loss of a stamen from the second whorl is clearly visible in A and B of Fig. 11. In C and D of Fig. 11 it can be observed the developing nectary tissue swells into the position of the missing stamen. In E and F of Fig. 11 it can be seen that the first whorl of stamens is positioned inside the second whorl, indicating obdiplostemony. The obdiplostemony observed for the stamen whorls does support the centrifugal initiation of these organs. Floral development of Cuphea (Lythraceae): Understanding the origin of monosymmetry and the epicalyx in the flower Celina Barroca | August 2014

RBGE | University of Edinburgh - 33 - MSc Biodiversity and Taxonomy of Plants

Plate 2

Fig.11. C. caeciliae SEM figure plate

Floral development of Cuphea (Lythraceae): Understanding the origin of monosymmetry and the epicalyx in the flower Celina Barroca | August 2014

RBGE | University of Edinburgh - 34 - MSc Biodiversity and Taxonomy of Plants

Plate 3

Petals present or reduced

A. C. caeciliae reduced petals (P); Sepals lobes (Sp) alternating with calyx emergences (Ec) – petal remnants opposite the calyx emergences; B. Detail of C. caeciliae reduced petal in an earlier stage; C. C. procumbens petal presence, still curved inwards.

Key for images: Sp = Sepals; Ec = Epicalyx

According to Fig. 12, the petals alternate with calyx lobes, as observed in A and B. In some species, for example C. caeciliae, petals are reduced down to small pointy structures.

In C. procumbens (Fig. 12, C), petals are present and distinctly petal-like. Those shown in this figure are two of the four small petals on the abaxial side of the hypanthium. The upper two petals are considerably larger.

Floral development of Cuphea (Lythraceae): Understanding the origin of monosymmetry and the epicalyx in the flower Celina Barroca | August 2014

RBGE | University of Edinburgh - 35 - MSc Biodiversity and Taxonomy of Plants

Plate 3

Fig.12. C. caeciliae and C. procumbens SEM figure plate

Floral development of Cuphea (Lythraceae): Understanding the origin of monosymmetry and the epicalyx in the flower Celina Barroca | August 2014

RBGE | University of Edinburgh - 36 - MSc Biodiversity and Taxonomy of Plants

Monosymmetry

Stamen plate:

A. Cuphea micropetala;

B. C. caeciliae;

C. C. hyssopifolia;

D. C. cyanea;

E. C. ignea;

F. C. procumbens.

In Fig. 13 (A – F), two levels of stamen insertion can be observed in all species mentioned; however the filaments are grouped into three different lengths (Fig. 13, B, D and E). Stamens are inserted along the abaxial side of the hypanthium. The length of the style varies and also tends to run along the abaxial half of the floral tube.

Floral development of Cuphea (Lythraceae): Understanding the origin of monosymmetry and the epicalyx in the flower Celina Barroca | August 2014

RBGE | University of Edinburgh - 37 - MSc Biodiversity and Taxonomy of Plants

*

*

* *

*

*

Fig. 13. Stamens of fixed dissected half mature flowers plate: stamen insertion levels and filament relative length of six species mentioned before. All observed at x10 magnification, except for C which was x40. * indicates the abaxial side of the flower.

Floral development of Cuphea (Lythraceae): Understanding the origin of monosymmetry and the epicalyx in the flower Celina Barroca | August 2014

RBGE | University of Edinburgh - 38 - MSc Biodiversity and Taxonomy of Plants

Nectary Plate:

A. Cuphea micropetala;

B. C. caeciliae;

C. C. hyssopifolia;

D. C. cyanea;

E. C. ignea;

F. C. procumbens

As it can be observed in Fig.14 (A – F), nectary shape and si11e varies according to the specie which belongs.

Although there is a variance in size of the nectary between species, the nectary sac surrounding it is generally large. While gynoecium and androecium develop along the abaxial side of the flower tube, the nectary develops at the base of the carpels occupying the space formed by the loss of the sixth stamen of the second whorl; and expands into the adaxial side of the floral tube.

Floral development of Cuphea (Lythraceae): Understanding the origin of monosymmetry and the epicalyx in the flower Celina Barroca | August 2014

RBGE | University of Edinburgh - 39 - MSc Biodiversity and Taxonomy of Plants

Figure 14 – legend below.

Floral development of Cuphea (Lythraceae): Understanding the origin of monosymmetry and the epicalyx in the flower Celina Barroca | August 2014

RBGE | University of Edinburgh - 40 - MSc Biodiversity and Taxonomy of Plants

Fig. 14. Nectary of fixed dissected half mature flowers plate: by descending order of size, and shape of the nectary of each specie; Unfortunately, there is no scale, but all were observe at a relatively same magnificationof x10 and x40.

FIG.15. Floral diagram of Cuphea micropetala, showing the ovary and level of insertion of the stamens and perianth (adapted from Ronse De Craene, 2010)

Floral formula:

Suggested general floral formula:

Floral development of Cuphea (Lythraceae): Understanding the origin of monosymmetry and the epicalyx in the flower Celina Barroca | August 2014

RBGE | University of Edinburgh - 41 - MSc Biodiversity and Taxonomy of Plants

Discussion

Comparative analysis of different species

Thyrse and racemes with uniparous cymes are dominant types of inflorescence in Cuphea. One of the species, C. ignea, is a solitary flower. This can be the result of an initial compound cyme reduce to a solitary flower.

All six species of Cuphea treated in this study have presented an elongated coloured hypanthium with a calyx tube where the stamens are inserted along in two different levels on the abaxial side, relatively close to the top; a superior ovary is present in all species as well as a nectary.

C. hyssopifolia and C. procumbens have six pink to purple petals well developed and C. cyanea have just two reflexed petals on the adaxial surface with a different colouration that might perform as a nectary guide. In all the other species the petals are absent and instead they have a called reduced petal to small pointed remnants that might be related with the presence of nectary and colourful hypanthia and indicating that they are no longer needed to attract pollinators.

Epicalyx origin

An important structure present in the six species is a theoretical epicalyx (theoretical as it would be expected but it is not a true epicalyx).

Several definitions of epicalyx were given by different authors mentioned previously and the ones defining it as an organ itself with bract origin (e.g. Cadet, 1954) and stipule origin (Eichler, 1878) are not accepted in the case of Cuphea due to, for example, the absence of stipules within the genus in contrary to other genera in Lythraceae. In addition, theoretical

Floral development of Cuphea (Lythraceae): Understanding the origin of monosymmetry and the epicalyx in the flower Celina Barroca | August 2014

RBGE | University of Edinburgh - 42 - MSc Biodiversity and Taxonomy of Plants epicalyx lobes appears in later stages of ontogeny on the calyx tube and not visible in the floral apex.

According to SEM images, it shows that calyx primordia initiates simultaneous with six sepals, expanding a hypanthium. While these calyx lobes develop, they bend towards the floral apex originating some appendages alternating with the sepals, after the calyx organ whorl is already formed. These appendages are what can be called a theoretical epicalyx and are an interpretation, in function of a valvate aestivation (Troll, 1949), of a result of the bending consequences of the sepal lobes linked with the congenital fusion of the calyx (Mayr, 1969). They can be described as special growths at the top of the floral tube (Cadet, 1954), however no homology can be stated with other different organs of origin forming an epicalyx, such as stipules or bracts.

Mayr’s (1969) definition of commissural appendages (i.e. a commissure is the place where two things are joined) is the most acceptable definition of a theoretical epicalyx formation in Cuphea, supported by his commissural stigma explanation (they have been found in some species of Onagraceae – sister group of Lythraceae) and Troll’s (1949) work on Campanulaceae. Commissural appendages can be defined as an outgrowth of two joining points (sepals united into a tube).

Due to the complicated attempt to define epicalyx as a sui generis organ and the several theories already proposed for different groups of plants, is suggested that a review in the concept of epicalyx should be taken in account.

Evolution of the flower in Cuphea: monosymmetry, merism

Floral morphology and the study how it develops should include all sequences of morphological changes during ontogeny. Unfortunately, there is no data available for earlier inception stages.

Floral development of Cuphea (Lythraceae): Understanding the origin of monosymmetry and the epicalyx in the flower Celina Barroca | August 2014

RBGE | University of Edinburgh - 43 - MSc Biodiversity and Taxonomy of Plants

The ontogeny development sequence in Cuphea is:

Calyx – Androecium – Gynoecium – Corolla (when present)

According to SEM analysis, the androecium initiates with two stamen whorls straight followed by the gynoecium. A centrifugal initiation is assumed due to a lack of initial stages information in the ontogeny, although is supported by obdiplostemony observed for the stamens whorls – the first whorl of stamens arise (six stamens, inner whorl) inside the second whorl (five stamens, outer whorl). A loss of a stamen from the second whorl may be explained through the necessity of an empty space for the developing of nectary tissue into that position.

Moreover, while gynoecium and androecium develop along the abaxial side of the flower tube, the nectary develops at the base of the carpels occupying the space formed by the loss of the sixth stamen of the second whorl; and expands into the adaxial side of the floral tube while the stamens and carpels move to the abaxial side, clearing the space for the pollinator to get its reward. The symmetry of the flower bud starts to change from radially symmetric to a dissymmetric calyx in early stages, ending in zygomorphic mature flowers.

Monosymmetry is driven by the formation of the large nectar “sac” in just one side (adaxial side), leading to the other organ whorls be pushed according to it, losing a stamen to accommodate it leading to zygomorphy to be expressed by differential growth.

Cladistic analyses (Tobe et al., 1998) indicate that the major floral characters of Cuphea can be derived like the globular nectary, the absence of petals and the zygomorphic symmetry.

Graham (2006) mention that petals are lost in several red, large-flowered species – the enhance colour of floral tubes presumably replaces petals as a pollinator attractant. The same might happen with the development of a nectary and absence of petal.

For example, a coloured hypanthia with petal size reduced plus presence of a nectary is an adaptation to bird pollination (e.g. C. micropetala) while species with showy and bright coloured petals are adapted to insect pollinator (e.g. C. hyssopifolia – bright pink petals – might be pollinated by bees while C. procumbens – bright purple petals – by a bigger insect, such as a butterfly, perhaps).

Floral development of Cuphea (Lythraceae): Understanding the origin of monosymmetry and the epicalyx in the flower Celina Barroca | August 2014

RBGE | University of Edinburgh - 44 - MSc Biodiversity and Taxonomy of Plants

Petals reduction, development of a nectary and changes in hypanthium sizes a response of pollination syndromes pressures, leading to the evolution of monosymmetry.

SEM technique is an essential tool for development studies and so comparative floral development is now being used as an approach to understanding the relationship between morphology, systematic and phylogeny and also the physiology with functional morphologic structures.

As Cuphea being designated a monophyletic genus, it is important to say that developmental studies in monophyletic lineages provide important information on the evolution of developmental patterns.

Floral development of Cuphea (Lythraceae): Understanding the origin of monosymmetry and the epicalyx in the flower Celina Barroca | August 2014

RBGE | University of Edinburgh - 45 - MSc Biodiversity and Taxonomy of Plants

Conclusions

Using the term epicalyx for all the structures with different origin referred previously is confusing and leads to incorrect conclusions. The large variety of epicalyces origins makes it difficult to come up with a comprehensive definition of the epicalyx structure.

Thus, it is accepted the interpretation of Mayr (1969) of commissural appendages result of the congenital fusion of the calyx linked to Troll’s (1949) explanation of commissural appendages of the sepals in function of the valvate aestivation.

Evolution of symmetry in Cuphea is probably largely driven by the pollinator syndromes within the genus, originated by the reduction or absence of petals and a development of a globular nectary.

Floral development of Cuphea (Lythraceae): Understanding the origin of monosymmetry and the epicalyx in the flower Celina Barroca | August 2014

RBGE | University of Edinburgh - 46 - MSc Biodiversity and Taxonomy of Plants

Recommendations

It would be likely to do a review for the epicalyx concept.

Limitations of techniques/methods/procedures during the research with the particular case of study – Cuphea were observed.

Paraffin embedding technique didn’t work as expected, suggesting Technovit for further studies.

Floral development of Cuphea (Lythraceae): Understanding the origin of monosymmetry and the epicalyx in the flower Celina Barroca | August 2014

RBGE | University of Edinburgh - 47 - MSc Biodiversity and Taxonomy of Plants

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Floral development of Cuphea (Lythraceae): Understanding the origin of monosymmetry and the epicalyx in the flower Celina Barroca | August 2014

RBGE | University of Edinburgh - 48 - MSc Biodiversity and Taxonomy of Plants

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Floral development of Cuphea (Lythraceae): Understanding the origin of monosymmetry and the epicalyx in the flower Celina Barroca | August 2014

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Floral development of Cuphea (Lythraceae): Understanding the origin of monosymmetry and the epicalyx in the flower Celina Barroca | August 2014