Is the flower resupinate or not?
The answer to this question is neither “yes” nor “no,” though flowers change their orientation during their development. As we observed using anatomical sections of an apical part of a flower-bearing shoot, the pedicel of a developing flower bud turns to a varying degree, in clockwise or anti-clockwise direction, according to the position of the leaf axils producing flower buds in relation to the ground surface. If the flower bud is borne from a leaf axil that is positioned horizontally, the pedicel turns 45° clockwise or anticlockwise (Fig. 2h). As a result, the flower is always positioned with its calceolus downward.
Why do the flowers of Emblingia change their orientation during their development? It is obviously associated with insect-pollination. By changing their orientation, the flowers have the anthers and ovary/stigma upward, the calceolus downward, and the nectary gland downward and deep inside. A combination of the anthers and stigma on the top of androgynophore is similar to the gynostemium in orchid flowers. Both the anthers and ovary/stigma are oriented inside the flower at anthesis (Fig. 3c). Because the flowers have fertile stamens only on the adaxial side with the staminodia (hood) on the abaxial side, they have the anthers posterior to the stigma (Fig. 3c). In addition, a large space like a tunnel develops between the androgynophore and calceolus (petals) of the flower. Although I did not see any insects on the plant in the field during the few hours in the morning of October 2009, it is possible that some insects had entered and exited the flowers after sucking nectar in the spur-like container deep in the flower. These insects probably crawled on or flew over the ground surface early in the morning or late in the evening because it is too hot in the daytime. When entering the flower, an insect provides the stigma with pollen grains from other flowers, and when leaving the flower, the insect gets pollen grains on its head or back from the anthers. The calceolus probably functions as a landing platform for insects.
Answers to the questions pertaining to the petals, stamens, nectary, hood, and carpels
Anatomical observations confirmed that two distinct petals which are placed downward at anthesis form the calceolus on the adaxial side. The two petals alternate with three of the five calyx-lobes or sepals, indicating that five petals existed originally; two of them developed into the calceolus and the remaining three became extinct. Melville in Erdtman et al. (1969, p. 178) assumed that “the hood represents a third petal which has become fused to the androgynophore in the course of evolution.” However, this is not likely because anatomical evidence shows that the hood is a morphologically complex structure comprising three to six connate staminodia (or sterile stamens), not representing a petal.
As for the questions of how many stamens originally existed and how many whorls of stamens are present in a flower, anatomical observations indicate that originally five stamens were present in a single whorl. As stated in the Introduction, Leins considered that ten stamens were originally present in two whorls of five stamens, whereas Melville assumed that five stamens originally existed in a flower. According to Melville’s interpretation, two latero-abaxial stamens are fertile, each dividing into two; two latero-adaxial stamens were lost and the abaxial stamen developed into a nectary gland. An anatomical examination of microtome sections revealed that five staminal traces are present in the lower levels of the androgynophore, indicating that the original number of stamens is five, and that they alternate with the calyx-lobes or sepals, rather than with the petals. The flowers of Emblingia are thus obhaplostemonous with double positions of the stamens and staminodia. Two of the five original stamens are fertile, and the three others sterile and staminodial. The vascular anatomy shows that every staminal trace upwardly bifurcates to supply two stamens on the same whorl, providing evidence for the occurrence of stamen dédoublement. Mueller (1860‒1861) and later authors always found four fertile stamens. Dédoublement, however, does not necessarily occur in all of the three sterile stamens. If it does not occur in one or two of the three sterile stamens, the flowers have four or five staminodes instead of six, as Melville observed as four or five “lobes” (Erdtman et al. 1969, p. 176). Melville interpreted one of the five stamens as having developed into a nectary gland, but this is incorrect. The nectary gland is positioned outside of the stamen whorl.
Finally, as for the number of carpels constituting a gynoecium, anatomical observations showed that the gynoecium is always three-loculed and formed by three carpels. Based on the 19 flowers that I examined anatomically, there were neither unicarpellate and unilocular gynoecium, as Melchior (1964) and Watson and Dallwitz (1992 onwards) describe, nor bicarpellate gynoecia. A single ovule is borne from an axial placentation in each locule, and the ovule never has a basal position in the locule as described by Takhtajan (1997, 2009) and Stevens (2001 onwards).
A summary of the floral morphology and structure of Emblingia
Prior to my analyses, information on floral morphology and structure of Emblingia calceoliflora has been controversial. As discussed above, anatomical and developmental analyses using fresh material have answered the questions stated in the Introduction and have updated some characters. The floral morphology and structure of E. calceoliflora can be summarized as follows.
Flowers solitary with a pedicel of about 4 mm long, pentamerous, and strongly monosymmetric. Calyx comprising five sepals, one median adaxial, two latero-adaxial, and two latero-abaxial. Sepals connate; a calyx tube apically dividing into five lobes, with a deep slit on the abaxial side in association with strong monosymmetry. Corolla comprising two latero-adaxial petals that alternate with three sepals on the adaxial and latero-adaxial side, developing into a large slipper-like form (calceolus) on the adaxial side. Two petals distinct throughout their length, basally valvate and apically imbricate. Two short longitudinal walls, both from the basal region of the petal, developing to form a spur-like container to enclose a swollen nectary gland. The nectary gland extrastaminal, adaxial, forming between the two petals.
Androecium originally comprising five stamens in a single whorl, which alternate with calyx-lobes or sepals and opposite the petals; thus, flowers obhaplostemonous. As shown in vascular anatomy, lateral dédoublement occurring in five original stamens, resulting in four fertile stamens on the adaxial side and (three to) six staminodia on the abaxial side. Fertile stamens bearing anthers above a short filament; staminodia lacking anthers, connate at the base to form the “hood.” Gynoecium tricarpellate, trilocular with a single ovule on axial placentation in each locule. Androgynophore long, with a combination of anthers and ovary/stigma like a gynostemium on the top. Both anthers and ovary/stigma turning inside into the flower at anthesis.
During flower development, a pedicel turning clockwise or anti-clockwise to various degrees according to flower-bud position, placing the calceolus downward and androgynophore upward in flowers at anthesis. Flowers at anthesis with a large space between the calceolus and the androgynophore and a nectary gland deep within it.
Comparisons with other families of the Brassicales
An understanding of the exact floral morphology and structure of Emblingia enables a comparison with flowers of the 17 other families of Brassicales. I reviewed as many characters of floral morphology and structure for the other families as possible using published data: Akaniaceae (Ronse de Craene et al. 2002), Bataceae (Ronse de Craene 2005), Capparaceae (Ronse de Craene and Smets 1997), Caricaceae (Ronse de Craene and Smets 1999), Gyrostemonaceae (Hufford 1996), Moringaceae (Olson 2003; Ronse de Craene et al. 1998), Pentadiplandraceae (Ronse de Craene 2002), Salvadoraceae (Ronse de Craene and Wanntorp 2009), Setchellanthaceae (Tobe et al. 1999), Tropaeolaceae (Ronse de Craene and Smets 2001). Thus all 17 families except for the poorly known Borthwickiaceae were considered (Ronse de Craene and Haston 2006). Ronse de Craene and Haston (2006) analyzed 42 characters relating to floral morphology, development and structure for cladistic analysis of Brassicales in their combined morphology-molecular study. Among these characters, six related to early floral development had no information for Emblingia. Although those six characters were also not resolved with the present study, data for the following six characters for Emblingia were revised. Flowers are monosymmetric (not polysymmetric) at maturity; androecial configuration is obhaplostemonous (not diplostemonous); staminodia occur in the same whorl as stamens (not in two whorls); reduction of stamen number within a whorl is absent; filaments are hairy (not glabrous); and the number of carpels is three (not three and two).
Based on all the available data, I mapped character states of more than 36 characters on a phylogenetic tree of the Brassicales using MacClade version 3.04 (Maddison and Maddison 2005). The tree was modified from Hall et al. (2004) and Su et al. (2012), placing Emblingiaceae as sister to the rest of the core Brassicales. Polarities of their respective character states were determined using the Neuradaceae, basalmost in the Malvales (Soltis et al. 2000), as an outgroup of the Brassicales, following a previous paper on Koeberliniaceae (Tobe and Raven 2008). Data on the Neuradaceae were obtained from Murbeck (1916), Ronse de Craene and Smets (1995), and unpublished data (Tobe, personal observation). In general the flowers are greatly diversified in the Brassicales, and homoplasies are frequent in many characters. Ultimately I selected six characters that are likely to represent floral evolution and diversity in Brassicales. Some of them have often been used to distinguish one group of families from another in the Brassicales (Ronse de Craene 2010). The characters are as follows: merism, symmetry, the presence or absence of a stalk supporting an ovary, position of nectaries, and placentation. The six characters (1–6) and their respective character states are presented in Table 1.
Figure 5a–f shows the distribution of character states of the six characters. While the flowers of Emblingia retain plesiomophies such as pentamery and axial placentation (Fig. 5a, f), they have apomorphies such as monosymmetry, androgynophore, obhaplostemony, and an extrastaminal nectary (Fig. 5b–e). Among the apomorphies, obhaplostemony is restricted to Emblingia, whereas monosymmetry and androgynophore are homoplasies that occur in a clade of Borthwickiaceae and Resedaceae. Monosymmetry is also found in Tropaeolaceae and Capparaceae pro parte, and androgynophore is also found in Setchellanthaceae. Only the extrastaminal nectary is a synapomorphy of Emblingiaceae and the rest of the core Brassicales [Borthwickiaceae (data uncertain), Brassicaceae, Capparaceae, Cleomaceae, Gyrostemonaceae Pentadiplandraceae, Resedaceae, and Tovariaceae], although it occurs as a homoplasy in Tropaeolaceae. Within the core Brassicales, the nectary is lost in Gyrostemonaceae (with anemophilous flowers).
Apart from the six characters discussed above, the presence of paired stamens is one of the noticeable features of Emblingia. The vascular anatomy of the androgynophore shows that the paired stamens receive their respective traces by bifurcation from a single stamen trace, providing evidence for the occurrence of stamen dédoublement. Such paired stamens are known in some other Brassicales, particularly in the core Brassicales. The flowers of Brassicaceae, Capparaceae, and Cleomaceae nearly always have two outer stamens and four inner stamens (Ronse de Craene 2010). Two pairs of the inner stamens have often been interpreted as a result of dédoublement, although this interpretation is doubted by Ronse de Craene and Smets (1993; references cited therein). I have reported that stamen chorisis (i.e., the splitting of a stamen into more than one) occurs in Setchellanthus (Setchellanthaceae) of the non-core Brassicales (Tobe et al. 1999). In Setchellanthus a total of 40‒70 stamens comprise six fascicles, each having been derived by stamen chorisis. Borthwickiaceae (Borthwickia trifoliata W.W. Smith only), a new monotypic, core-brassicalean family recently proposed, has 60‒70 stamens in a flower (Su et al. 2012). Some species of Capparaceae such as Boscia senegalensis Lam., Capparis cynophallophora L., C. micracantha DC., and Colicodendron yco (Mart.) Mart. (“Capparis yco”) are known to have as many as 40 stamens that are centrifugally initiated (Ronse de Craene and Smets 1997; Ronse de Craene 2010). Those stamens might have been derived by stamen chorisis as in Setchellanthus. To the best of my knowledge, the centrifugal stamen initiation is closely associated with stamen chorisis as in Glaucidium (Ranunculaceae) (Tamura 1972), Harungana (Hypericaceae) (Ronse de Craene and Smets 1991), and Paeonia (Paeoniaceae) (Hiepko 1964, 1966; Sawada 1971). The occurrence of dédoublement in Emblingia suggests that stamen splitting (chorisis and dédoublement) might be a generalized feature in the Brassicales, at least in the core Brassicales.
Overall, the flowers of Emblingia have a distinctive combination of plesiomorphies and apomorphies [synapomorphies (including homoplasies) and autapomorphies). Based on the available information, the extrastaminal nectary is the only synapomorphy supporting the core-Brassicales including Emblingiaceae, although it is uncertain in Borthwickiaceae. More strictly, however, the nectary gland of Emblingia differs from those in the other core-brassicalean families because it is enclosed by the spur-like container developed from petals in the bottom of the flower. Many of the floral features of Emblingia represent a unique syndrome of insect pollination. Emblingia seems to have survived in close association with the pollinators adapted to the particularly dry habitat of Western Australia.