Floral development in Impatiens chishuiensis (Balsaminaceae): formation of two well-developed anterolateral sepals and four carpels

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1 Plant Syst Evol (2010) 286:21 26 DOI /s ORIGINAL ARTICLE Floral development in Impatiens chishuiensis (Balsaminaceae): formation of two well-developed anterolateral sepals and four carpels S. X. Yu X.-R. Zhou G.-F. Xu L. Meng H.-Y. Bi Received: 16 December 2008 / Accepted: 19 February 2010 / Published online: 28 March 2010 Ó Springer-Verlag 2010 Abstract Impatiens is a highly diversified genus with remarkable zygomorphic flowers. Floral ontogeny of three species of Impatiens has recently been studied, which suggested that, during the course of evolution, the anterolateral sepals become reduced and that these rudimentary sepals fuse postgenitally or congenitally with the anterior petal. All three belong to the majority type with five carpels and no mature anterolateral sepals. However, a few species of Impatiens have four carpels and two well-developed mature anterolateral sepals, and their floral development has never been examined. From a comparative-ontogenetic viewpoint, we explore the mechanism of formation of welldeveloped anterolateral sepals and four carpels in I. chishuiensis with five sepals and four carpels. The results show that, if anterolateral sepals are present, their early development statuses will likely influence whether or not the sepals can fully develop later, and that the initiation and development of five stamens have an effect on the shapes of floral apexes, eventually resulting in congenital fusion of two adaxial carpels. Furthermore, we discuss the systematic and evolutional value of floral early development data. S. X. Yu Documentation and Information Center, Institute of Botany, Chinese Academy of Sciences, Beijing , China X.-R. Zhou (&) G.-F. Xu L. Meng Department of Life Science and Technology, Henan Institute of Science and Technology, Xinxiang , Henan, China xiuren_zhou@yahoo.com H.-Y. Bi Beijing Museum of Natural History, Beijing , China Keywords Floral development Anterolateral sepals Number of carpels Shape of floral apex Impatiens chishuiensis Introduction The genus Impatiens L. with about 1,000 species and the monotypic genus Hydrocera B. together constitute Balsaminaceae (Grey-Wilson 1980; Fischer 2004). H. triflora (L.) Wight et Arn., a semi-aquatic herb native to the Indo- Malesian countries, can be readily distinguished from the species of Impatiens by five free petals and indehiscent berry-like fruit. Impatiens is a highly diversified genus, occurring mainly in montane regions in the Old World tropics and subtropics (Grey-Wilson 1989; Fischer and Rahelivololona 2002; Fischer 2004). The precise developmental relationships among the different floral parts are complex, and blurred by the upside-down growth (resupination) of the flowers and the use of different descriptive terms for the position of floral parts. Our description and discussion about floral parts and ontogeny follow the terminology of Caris et al. (2006). The genus Impatiens has remarkable zygomorphic flowers which have three (rarely five) free sepals. Generally, the lower sepal (posterior sepal or spurred sepal) is large, petaloid, and constricted into a nectariferous spur. The corolla consists of five petals: an upper one (anterior petal or standard), which is flat or cucullate, sepaloid; and four lateral ones, which are united in pairs (wings). The five stamens have short and flat filaments with a scale-like appendage inside, incompletely capping the gynoecium. The gynoecium has five or occasionally four carpels forming a syncarpous superior five- or four-loculed ovary with a very short style or no style at all. The fruit is a

2 22 S. X. Yu et al. five- or four-valved loculicidal fleshy capsule, usually dehiscing explosively (Grey-Wilson 1980; Fischer 2004; Chen et al. 2008; Yu 2008). The current classification, mainly based on floral morphology, is impractical, because the fragile nature of the flowers makes identification of dried specimens even to section or subgenus difficult or impossible. It has also turned out to be unnatural (Yuan et al. 2004; Caris et al. 2006). Recently, robust molecular phylogenies have been obtained. However, except for spurless flowers and broadly fusiform capsules, which are revealed to be two synapomorphies for two different DNA-based clades, respectively, almost no other morphological synapomorphy for DNA-based clades was identified (Yuan et al. 2004; Janssens et al. 2006a, 2006b), which is necessary for the construction of a working classification (Soltis et al. 2005). Floral ontogeny may help to provide taxonomically useful characters and identify more morphological or developmental synapomorphies for DNA-based clades (Caris et al. 2006). Floral ontogeny has recently been studied in three species of Impatiens: I. niamniamensis Gilg, I. columbaria J.J. Bos, and I. hawkeri W. Bull (Caris et al. 2006). It was suggested that, during the course of evolution, the anterolateral sepals become reduced, and that these rudimentary sepals fuse postgenitally or congenitally with the anterior petal. According to Caris et al. (2006), a flower with three sepals is derived from one with five sepals. Moreover, it is shown that the outer part of the septa plays a key role in explosive dehiscence of the capsules. They described and discussed floral development and evolution in three species which all belong to the majority type with three sepals (no anterolateral sepal) and five carpels. However, a few species of Impatiens have five mature sepals (including two well-developed anterolateral sepal) and four carpels (Shimizu and Takao 1985, 1982; Chen et al. 2008), and how the flower structures are produced is unknown or undiscussed. To examine the ontogeny of flowers with five sepals and four carpels and explore the mechanism of formation of well-developed anterolateral sepals and four carpels, we have studied I. chishuiensis Y.X. Xiong from Guizhou Province of China. Materials and methods The material used in the study was obtained from a wild population in Chishui, Guizhou, China. Voucher specimens (S. X. Yu 4065, GUIZHOU) are deposited in the China National Herbarium (PE). The material was fixed in 40% formalin, acetic acid, 70% alcohol (FAA; 5:5:90). Floral buds were dissected in 80% ethanol under a Nikon-2100 stereomicroscope equipped with a cold light source. The material was then dehydrated through an ethanol series, ethanol-isopentylisopentanoate mixture, and 100% isopentyl-isopentanoate, dried in a Dento DCP-1 apparatus using liquid CO 2, and placed on aluminum stubs with agglutinant. They were then coated with gold palladium in a Technics Hummer V sputter coater. Micrographs were taken with a Hitachi S-4800 scanning electron microscope (SEM) at 10 kv (Zhou et al. 2008). Results In I. chishuiensis, there are 2 11 flowers, each with a subtending bract, in each raceme with a 4- to 11-cm-long peduncle in the axils of the leaves (Fig. 1a). The slender pedicels are cm long. The flowers are cream and have four carpels and five sepals, and the two anterolateral sepals are well developed (Fig. 1a, b, d). The pale-yellow lower sepal is broadly funnel shaped, 2 cm long, with a 1.5- to 1.8-cm-long spur (Fig. 1c). The floral primordia are spirally initiated along the axis (Fig. 2a). The early flower meristems emerge as an elliptical mound between the base of the bract and the apical meristem and are completely covered and protected by the bract primordium (note that the bracts have been removed in all figures). Then, simultaneously with further uplift of the flower primordium, the first two sepals (posterolateral sepals) originate from both right and left sides (Fig. 2a, b). As the two lateral sepals grow, the apical domain between them becomes more massive (broad) (Fig. 2c). Then, a posterior sepal primordium (the spurred sepal) and two abaxial ones (anterolateral sepals) are almost synchronously initiated at Fig. 1 Inflorescence and mature flowers of I. chishuiensis. a Inflorescence, showing a typical raceme. b Flower, top view showing two well-developed anterolateral sepals and two posterolateral sepals. c Flower, lateral view showing a broadly funnel-shaped petaloid sepal with a long spur. d Flower, frontal view showing the androecium covering the gynoecium. (Bar a d = 8 mm)

3 Floral development in Impatiens 23 Fig. 2 Early morphological development of I. chishuiensis flowers. a Top-view SEM of an inflorescence apex indicating floral primordia initiated spirally. b Initiation of the two lateral sepals. c The expanding region between left and right sepals. d Initiation of anterolateral and posterior sepals on abaxial and adaxial sides, respectively, showing that they arise almost simultaneously. e f Further growth of anterolateral and posterior sepals, and expansion of undifferentiated top region. g Initiation of anterior petal (arrowhead) between both anterolateral sepals. h Origin of the rest of petals after the initiation of the anterior petal. i Growth of five petals showing a remarkable gap (arrow) between posterolateral petals. j Synchronous initiation of five stamens with the adaxial stamen close to the gap between both posterolateral petals. k l Further development of stamens showing different growth rates of different stamens and an undifferentiated floral apex formed like a wave-like irregular pentagon. Bracts have been removed in all figures. Lateral sepals have been removed in c and e g. All sepals have been removed in h and i. All sepals and anterior petal have been removed in k and l. All sepals and petals have been removed in l. Asterisk indicates apex of inflorescence. Adaxial sides are on the top in b l. Contrast was adjusted by using Photoshop 7.0. (FP floral primordium, B bract, S sepal, P petal, A stamen) (Bar a, d l = 40 lm, c d = 20 lm) adaxial and abaxial sides, respectively (Fig. 2d). At the early stages, the spurred sepal grows slightly faster than the two anterolateral sepals (Fig. 2d g). When these three sepals appear triangular and grow inwards after a longer duration of development and growth, the abaxial petal primordium (anterior petal) has arisen like a hemispherical mound between the two anterolateral sepals (Fig. 2g, h). Subsequently, the floral apex becomes slightly elongated and expanded; next the four lateral petal primordia are initiated and grow out independently (Fig. 2h, i). The growth of the posterolateral ones (upper pair of petals) is slightly faster than that of the anterolateral ones (middle pair of petals); however, the anterior petal is always the biggest one (Fig. 2i, j). It is worth noting that there is an obvious gap between the posterolateral petals (Fig. 2i, j). At the early stages, each pair of lateral petals become fused at their bases (Fig. 2k). Five stamen primordia arise simultaneously in an inner whorl, alternating with the petal

4 24 S. X. Yu et al. Fig. 3 Morphological development of I. chishuiensis gynoecium. a Undifferentiated top among five stamens showing an irregular wave-like pentagon. b Initiation of carpels showing that four carpels arise simultaneously and that the adaxial carpel is much larger than the others. c e Further growth of carpels showing that the adaxial carpel is still larger than the other carpels. f g Formation of ovary showing that carpels are closed into one ovary and that the adaxial carpel becomes of the same size as the other carpels. h Stigma showing four undifferentiated lobes. i Stamen appendages capping the ovary. Adaxial sides are on the top in a h. Contrast was adjusted by using Photoshop 7.0. (A stamen, AC adaxial carpel, C carpel, SA stamen appendage) (Bar a b, h = 20 lm, c d = 40 lm, f = 80 lm, g, i = 100 lm) (primordia) (Fig. 2j). However, the androecium soon becomes zygomorphic because of different growth rates of the stamen primordia. What is more, the two lower stamens seem to be closer to adaxial direction (Fig. 2k, l). It is interesting that, since the upper stamen of I. chishuiensis is close to the gap between the posterolateral petals, the shape of floral apex (an apical undifferentiated region) looks like an irregular wave-like pentagon with a short adaxial edge, two longer lateral ones, and two intermediate lower edges (Figs. 2k, l, 3a). Subsequently, four carpel primordia are formed simultaneously (Fig. 3b). Only one carpel is initiated in the adaxial region (Fig. 3b, c). At an early stage of carpel development, the adaxial carpel primordium is significantly larger than the other three (Fig. 3b e). Along with the growth and development of the carpels, their bases soon fuse with each other (Fig. 3c, d). The adaxial carpel gradually becomes the same size as any one of other three carpels (Fig. 3f, g). Meanwhile, the carpellary flanks curve and grow inward, resulting in closure of the ovary top (Fig. 3e g). The entire gynoecium looks like a square barrel (Fig. 3g). Style and stigma development have not yet taken place (Fig. 3h, i). The fused five stamen appendages incompletely cap the gynoecium, and the adaxial one of them is clearly pushed backward (Fig. 3i). Discussion If anterolateral sepals are present, the level of their early development will influence whether or not the sepals can fully develop. Although, in the early development of the flower, two anterolateral sepal primordia occur in both I. chishuiensis and I. columbaria, mature flowers have well-developed anterolateral sepals in the former but not in the latter (Caris et al. 2006). Comparing the early floral development of the two species, the early development level of two anterolateral sepals in I. chishuiensis is significantly stronger than that in I. columbaria, in that the anterolateral sepal primordia in the former are almost the same size as other ones, whereas their size in the latter are remarkably smaller than the others. Concomitant with floral ontogenesis, it is clear that the anterolateral sepals of I. columbaria become rudimentary and finally disappear because of postgenital fusion with the anterior petal (Caris et al. 2006); on the contrary, the sepals of I. chishuiensis fully develop. The opposite destiny of anterolateral sepals between I. chishuiensis and I. columbaria may result from their different early development levels. In I. chishuiensis, the

5 Floral development in Impatiens 25 Fig. 4 Diagrammatic comparisons of organ positions and initiation from I. chishuiensis, I. columbaria, I. niamniamensis, and I. hawkeri before flower resupination. a I. chishuiensis, showing the early strong development of anterolateral sepals and the floral apex between stamens which results in congenital fusion of two adaxial carpels. b I. columbaria, showing that the early development of two anterolateral sepals is very weak, finally resulting in postgenital fusion. c I. niamniamensis, showing no observable initiation of anterolateral sepals. d I. hawkeri, showing no observable initiation of anterolateral sepals. In I. chishuiensis, the floral apex between five stamens forms an irregular wave-like pentagon; however, in I. columbaria, I. niamniamensis, and I. hawkeri, it displays a regular wavelike pentagon. b d are all drawn after Caris et al. (2006). Numbers indicate the order of organ initiation, and organs marked by the same number indicate almost synchronous initiation. Letters represent the types of organs (S sepal, P petal, St stamen). Central black solid circles or ellipse indicate carpels. Top black dots represent the axis bearing the flowers, and the gray crescents in bottom represent subtending bracts. Adaxial sides are on the top in a d. Diagrams were drawn and adjusted by AutoCAD 2004 and Photoshop 7.0 stronger early development of anterolateral sepals likely extends their development duration and maintains their larger volume, resulting in the formation of two mature anterolateral sepals; however, in I. columbaria, the weaker early development of anterolateral sepals probably shortens their development duration and reduces their original size, which finally leads to postgenital fusion between the sepals and anterior petal (Caris et al. 2006). It might be that, if anterolateral sepals are present, their development status likely plays an important role in influencing whether or not the anterolateral sepals can fully develop. Floral apex shape plays a crucial role in determining the number of carpels in I. chishuiensis. During the course of floral development, the centers of activity of growth and differentiation are initiated and subsequently replaced by other ones at other sites, and each center influences its neighborhood, in that the adjacent regions have to adjust to the newly arisen changes (Endress 1996). In the ontogeny of I. chishuiensis, the floral apex between five stamens is significantly different from the species studied by Caris et al. (2006) in that it is an irregular wave-like pentagon (Fig. 4a d). It is likely that well-developed anterolateral sepals influence initiation position and development of the corolla whorl (in particular anterior and anterolateral petals) and sequentially impact on the initiation position and development of stamens. Moreover, organogenesis also follows a principle that new primordia appear in the largest free space available (Hofmeister 1868; Leins 1964; Endress 1996). Possibly because of the larger free space, the adaxial stamen of I. chishuiensis is initiated at the upper region near to the gap between the posterior petals (Fig. 2j, k), so the floral apex (the central, undifferentiated region), where carpels will arise, forms an irregular wave-like pentagon in which the adaxial edge is shortened, the two middle ones are prolonged, and the two lower ones are intermediate in comparison with the other three species investigated by Caris et al. (2006) (Figs. 2j, l; 4a). At an early stage of carpel development, the one adaxial carpel is significantly larger than the other three carpels, and concomitant with development and growth, the adaxial carpel gradually become of the same size as the other three (Fig. 3b g). It is likely that the narrow adaxial region of the floral apex could not provide enough space for visible independent initiation of two adaxial carpels, resulting in the case that the initiation positions of the two carpels are close to or even overlap each other. In I. chishuiensis, therefore, it is suggested that the two adaxial carpels likely fuse congenitally, which might be similar to congenital fusion between anterior petal and anterolateral sepals in I. niamniamensis and I. hawkeri (Caris et al. 2006). The floral ontogeny of I. chishuiensis suggests that the initiation and development statuses of five stamens can influence floral apex shape and eventually have an effect on carpel initiation and development. Therefore, it is floral apex shape that plays a crucial role in determining the number of carpels in Impatiens. Early floral development provides some possible systematic and evolutional information in Impatiens. Although both I. chishuiensis and I. columbaria have anterolateral sepal primordia in floral early development (Fig. 4a, b), the sepals fully develop in the former but postgenitally fuse with anterior petal in the latter, likely because of different early development statuses (Caris

6 26 S. X. Yu et al. et al. 2006). Unlike the two species above, anterolateral sepals are not initiated visibly in I. niamniamensis and I. hawkeri owing to the congenital fusion between anterolateral sepals and anterior petal (Fig. 4c, d). Moreover, it is interesting that the anterior petal arises significantly earlier than other petals in the former, whereas all five petals originate simultaneously in the latter (Fig. 4c, d) (Caris et al. 2006). Consequently, in spite of the fact that congenital fusion of anterolateral sepals and anterior petals occurs in both I. niamniamensis and I. hawkeri, the mechanism of fusion becomes more likely diversified. In addition, three species (I. niamniamensis, I. columbaria, and I. hawkeri) without mature anterolateral sepals have five carpel primordia in a floral apex with a regular wavelike pentagon shape (Fig. 4b d) (Caris et al. 2006); in contrast I. chishuiensis, with two well-developed mature anterolateral sepals, produces four carpels in one with an irregular wave-like pentagons shape (Fig. 4a). Therefore, different development patterns would cause different mature character; however, same superficial features of mature organs may occur due to convergence in divergent taxonomic groups (Peréz and Vargas 2003; Endress 1996). In Impatiens, it is possible that the early floral development patterns provide some significant information on systematics. Some Impatiens species with five sepals and four carpels, such as I. calcicola, I. mirabilis, and I. saraburiensis, have been examined anatomically (Shimizu and Takao 1982, 1985). However, these species can be distinguished from the group including I. omeiana, I. chishuiensis, etc. by postgenitally united wing petals (Shimizu and Takao 1982, 1985; Lu 1991; Yu 2008). Molecular studies have proven that I. omeiana, I. auriculata, I. wilsonii, etc. from China, which have four carpels and five sepals like I. chishuiensis, form the most basal monophyletic group in the genus Impatiens (Yuan et al. 2004; Yu 2008). Therefore, in Impatiens, it is possible that two independent lineages from a common completely pentamerous ancestor have developed: one with a reduction in the number of carpels, and one with a reduction in the number of sepals. Floral morphology includes not only the mature floral structure but also the succession of morphological changes which can be seen during ontogeny, and comparative floral development is used as an approach to understand the relationship between morphology and phylogeny (Leins et al. 1988a, 1988b). Early floral development may provide useful information about systematics and phylogeny (Caris et al. 2006). Although the morphology and structure of mature Impatiens flowers are extremely diverse, they have little systematic and phylogenetic value, possibly because of convergence. This study shows that early development can provide some possible significant information about systematics and evolution of Impatiens. Acknowledgments We thank Magnus Liden, Yi-Lin Chen for their help and critical comments on the manuscript. References Caris PL, Geuten KP, Janssens SB, Smets EF (2006) Floral development in three species of Impatiens (Balsaminaceae). Am J Bot 93(1):1 14 Chen YL, Akiyama S, Ohba H (2008) Balsaminaceae. In: Wu ZY, Raven PH (eds) Flora of China, vol 12. Missouri Botanical Garden, St. Louis, pp Endress PK (1996) Diversity and evolutionary biology of tropical flowers. Cambridge University Press, Cambridge Fischer E (2004) Balsaminaceae. 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