Floral ontogeny of Tachigali (Caesalpinioideae, Fabaceae) species

Introduction

The Fabaceae family is one of the largest angiosperm families and figures among the most relevant groups for its economic and ecological importance (LPWG, 2017). This family is well known for its floral morphology, in particular its wide variety of shapes, as observed not only in the characteristics papilionaceous flowers, but also in actinomorphic, or asymmetric flowers observed among the group (Tucker, 2003). Studies of floral development have been helping the understanding of such morphological varieties and their homologies among Fabaceae flowers (Bruneau et al., 2014; Leite, Mansano & Teixeira, 2014; Leite et al., 2015; Prenner et al., 2015; Prenner & Cardoso, 2017). However, in Caesalpinioideae, a subfamily with a great variety in terms of floral architecture, many of its groups are poorly documented regarding floral ontogeny and development, mainly after its new circumscription (Tucker, Stein & Derstine, 1985; Tucker, 1988; Tucker, 1991; Tucker, 1992a; Tucker, 1996; Tucker & Kantz, 1997; Prenner, 2004; Marazzi & Endress, 2008), as seen for Tachigali Aubl. (Caesalpinioideae) (Casanova et al., 2020).

Tachigali is an important genus among the Neotropical arboreal leguminous flora (Silva & Lima, 2007), mainly owing to its ecological relevance as one of the most abundant genera of the Amazon rainforest (ter Steege et al., 2013). Within Arapatiella Rizzini & A. Mattos and Jacqueshuberia Ducke they form the Tachigali clade (Haston, Lewis & Hawkins, 2005). Most recently, the genus Sclerolobium Vogel has been circumscribed to Tachigali based on studies of wood morphology (Barreta-Kuipers, 1981; Macedo et al., 2014), pollen grain morphology (Graham & Barker, 1981) and external morphology of species (Silva & Lima, 2007; van Der Werff, 2008). According to Silva (2007), Tachigali genus covers tree species with simple or compound stipules paripinnate leaves. The inflorescences are in panicles with deciduous bracts. Its flowers vary from actinomorphic to zygomorphic, sessile to pedicellate, with cupular or tubular receptacle; all with five sepals, five petals and eight to 10 stamens. The gynoecium is stipitate, with the stipe attached to the center of the hypanthium or displaced laterally. Its fruits are of the cryptosamara type.

As a consequence of its circumscription, the variety of floral morphology within Tachigali has been expanded, currently including species with both actinomorphic and zygomorphic flowers with spatulated and/or filiform petals, tubular or cupular-shaped hypanthium, and ovary stipe placed in a central position, or laterally dislocated, in the hypanthium (van Der Werff, 2008), all of which has created some difficulty in understanding the relationships among the species within the group. Despite this wide variation, Casanova et al. (2020) in a recent work demonstrated that some aspects, such as unequal development of the hypanthium, are crucial for its floral symmetry which shows that their flowers are zygomorphic before anthesis and alternate between actinomorphic and zygomorphic after anthesis, owing to unique petals and stamens.

Comparative studies of external morphology and anatomy have helped us to understand the genus circumscription (Casanova et al., 2020); nonetheless, ontogenetic studies can shed new light by the addition of novel characters and, thereby, strengthen knowledge about the flower morphological diversity within Tachigali and among Caesalpinioideae (Tucker, 1992b; Tucker, 2003; Bruneau et al., 2014; Leite, Mansano & Teixeira, 2014; Leite et al., 2015; Prenner et al., 2015; Prenner & Cardoso, 2017). Therefore, the present work aims to perform an ontogenetic study of T. denudata, T. paratyensis and T. spathulipetala, three morphologically representative species from the Tachigali, which includes small and large flowers, variable in morphological terms. Tachigali paratyensis presents zygomorphic flowers, long pedicellate, with tubular hypanthium, spatulate petals, heteromorphic stamens and ovary stipe attached laterally to the hypanthium wall. Tachigali spathulipetala has actinomorphic flowers, also pedicellate, with cupular hypanthium, heteromorphic petals (spatulate and filiform), isomorphic stamens, and ovary stipe attached laterally to the hypanthium wall. Tachigali denudata has actinomorphic flowers, sessile to short pedicellate, with cupular hypanthium, filiform petals, isomorphic stamens, and ovary stipe in a central position in the hypanthium (Silva 2007 and Silva et al., 2016). These ontogenetical studies will promote a better understanding of the genus floral organs varieties, their symmetry, and homologies among Caesalpinoideae.

Materials and Methods

Three species of the genus Tachigali, Tachigali denudata (Vogel) Oliveira-Filho, T. paratyensis (Vell.) H. C. Lima, and T. spathulipetala L. G. Silva, L. J. T. Cardoso, D. B. O. S. Cardoso & H. C. Lima, were chosen to study the floral ontogeny as they represent the floral morphology variation observed in the genus: small and large flowers with actinomorphic/zygomorphic symmetry, spathulate and/or linear petals, the ovary stipe arising in a central portion or laterally displaced in the hypanthium. Flower buds of T. paratyensis (RB 43559) were collected at the Arboretum of the Rio de Janeiro Botanical Garden and fixed in glutaraldehyde 2.5% in 0.1 M sodium phosphate buffer at pH 7.2 (Gabriel, 1982). Tachigali spathulipetala samples (RB 460994; RB spirit 1327; RB 459830; RB spirit 1329) were obtained from the Spirit Collection of the herbarium from the Rio de Janeiro Botanical Garden and fixed in ethanol 70%. Tachigali denudata (RB 659134) samples were taken from the personal collection of Professor Haroldo C. Lima, which were also preserved in ethanol 70%. Flower buds at different developmental stages were dissected with the Leica MZ16 stereomicroscope.

The material was then dehydrated in ethanolic series and, subsequently, in acetone (Bozzola & Russel, 1999, modified). Dehydration was concluded using the Bal-Tec Critical Point Dryer CPD 030. Samples were then fixed with double-sided carbon adhesive tapes to stubs and then covered with a thin layer of gold (~20 nm), using the Emitech K550X Sputter Coater. The material was analyzed with the Zeiss EVO 40 and JEOL JSM-6390LV scanning electron microscope.

To describe the type of aestivation, we followed the Endress (1994) classification.

Results

All analyzed species have paniculated inflorescence. Flowers and flower buds develop acropetally and helically along the inflorescence axis (Fig. 1). In the apical zone of the inflorescence, a bract protects each flower bud. The bracts develop before the floral primordium, covering the buds exclusively during the early stages, senescing as they mature and approach floral anthesis. The flower buds follow the basic pentameric plan of leguminous plants, containing five sepals, five petals, five stamens in a double whorl and one carpel. Because of differences detected in the developing patterns of sepals and petals (Table 1), results related to the ontogeny of the floral whorls were divided into two patterns.

Table 1:

Summary of floral ontogeny results of the analysed species.

Species	Shared characters	Differential characters	Floral diagram
Tachigali denudata	Sepals: helical development, quincuncial aestivation; first one adaxial. Petals: helical development, slightly cochlear aestivation; first one abaxial and fifth adaxial. Androecium: unidirectional development. Gynoecium: adaxial carpel cleft, adaxial style bend.	Sepal: second abaxial; third lateral; fourth adaxial and fifth lateral; late trichomes. Petal: third abaxial and fourth lateral; filiform; early trichomes on the abaxial surface and on the carpel.
Tachigali paratyensis		Sepal: second adaxial, third and fourth in lateral position; fifth adaxial; early trichomes on the adaxial surface. Petal: third in lateral position and fourth abaxial; heteromorph (spatulate); late trichomes on the abaxial surface and on the carpel.
Tachigali spathulipetala		Sepal: second abaxial; third in lateral position and fourth adaxial; late trichomes in the sepals. Petal: third abaxial and fourth in lateral position; heteromorph (linear-lanceolate, spatulate); early trichomes on both surfaces and on the carpel.

DOI: 10.7717/peerj.13975/table-1

In the first pattern, sepal development of T. paratyensis follows a helical pattern (Figs. 2A–2D). The first sepal is formed in the abaxial region close to the bract (Fig. 2A), followed by the second sepal in the adaxial region close to the inflorescence axis (Fig. 2B). The third sepal appears in a lateral position (Fig. 2B), as well as the fourth, which originates in the sequence (Fig. 2C); finally, the last sepal also appears in the adaxial region (Fig. 2D). In the next stage of development, it is evident that the first sepal, which was removed in Fig. 2E, has a completely outer position, as well as the second sepal (Figs. 2C–2E). They present a quincuncial aestivation type (Figs. 2D and 2E). At the beginning of sepal development, the formation of simple trichomes can be observed (Figs. 2B–2D). The petal development is helical (Figs. 2F and 2G; Figs. 3A and 3B). Similar to the sepal, the first petal is formed in the abaxial region (Fig. 2F), followed by the second sepal laterally placed and then by the third in the lateral opposite side (Fig. 2F). The fourth petal grows in the abaxial zone (Fig. 2F) and the fifth in the adaxial zone (Fig. 2F). The petals initiate their formation only after the setting of the five sepals (Figs. 2F and 2G) in an interspersed manner. The aestivation type is defined as cochlear (Figs. 3A and 3B). Moreover, it is important to highlight that both sepal and petal whorls might develop clockwise and counterclockwise (Fig. 2B).

In the androecium, the external whorl is formed by antesepalous stamens, which initiate their development soon after the beginning of petal formation (Figs. 2F and 2G). The antesepalous stamens follow a unidirectional pattern (Figs. 2F and 2G; Fig. 3A) with the first stamen in the abaxial region (Fig. 2F), followed by two stamens at a lateral position (Figs. 2F and 2G) and two in the adaxial region (Fig. 2G; Fig. 3A). After the setting of the outer whorl of the androecium, the formation of the inner whorl occurs (Fig. 3C). They present antepetalous stamens (Fig. 3C), which also follow a unidirectional pattern; however, two stamens appear in the abaxial region: two laterally placed and one in the adaxial region. Upon establishment of the carpel, the anthers begin to differentiate, first on the outer whorl of stamens and then on the inner whorl (Figs. 3D and 3E), establishing themselves at distinct levels (Fig. 3F). In the already differentiated anthers, it is possible to observe an extension of the dome-shaped connective.

The carpel initiates its development with the antesepalous stamens. The carpel cleft is adaxial, and it soon closes, curving towards the adaxial region (Figs. 3A–3D). In addition, the style prolongates towards the adaxial region, along with the differentiation of the anthers (Figs. 3E–3G).

In the second pattern, for both T. denudata (Fig. 4; Figs. 5E and 5F) and T. spathulipetala (Figs. 5A–5D), the sepals are formed in a modified helical pattern. The first sepal emerges in the abaxial region (Fig. 4A), and the second emerges beside the former, also in the abaxial region (Figs. 4A and 4B). The third sepal develops laterally, the fourth in the adaxial side of the flower bud, and, finally, the fifth is formed in the lateral opposite side (Fig. 4B). They present a quincuncial aestivation type (Fig. 4C), and the trichomes only begin their development after settling of the sepals is concluded (Fig. 4C).

Petal formation also presents a helical pattern interspersed with the sepals (Fig. 4D). The first petal develops in the abaxial region of the flower bud, followed by the second laterally placed, the third in the abaxial region, the fourth also laterally, opposite to the second, and, finally, the fifth petal in the abaxial region (Fig. 4D). All T. denudata petals present a filiform shape (Figs. 4E and 4F), and don’t keep contact with each other, i.e., the imbricate aestivation is not present. In contrast, T. spathulipetala exhibits divergence in the form of petals. For some specimens, only the fourth and fifth petals become filiform (Fig. 5A), while for others, exclusively the fifth (Fig. 5B). Petal aestivation type is cochlear (Fig. 5B). For some individuals of T. spathulipetala, the fifth petal overlaps the fourth (Fig. 5A). In both species, petals present simple trichomes on both abaxial and adaxial sides (Fig. 5B).

In the androecium, the double whorl of stamens develops in a manner similar to that of T. paratyensis in a unidirectional pattern (Fig. 4D; Fig. 5C). The anthers of the antesepalous stamens begin their expansion before the closure of the carpel cleft, followed by the anthers of the antepetalous stamens (Fig. 5D). At the mature anthers, it is possible to detect an expansion of the dome-shaped connective, which is more apparent in T. denudata (Fig. 5E). The carpel begins its formation, together with the antesepalous stamens (Fig. 4D), and the carpel cleft is adaxial (Fig. 4E), as also observed for T. paratyensis. After its closure, the carpel starts to expand and curve towards the adaxial region of the flower bud (Figs. 5E and 5F). Simple trichomes are observed throughout the carpel (Fig. 4F; Figs. 5C–5F).

Discussion

Conclusions

This study is an unprecedented record of ontogeny in Tachigali. We found important characteristics in floral development of the species, such as sepals and petals with helical development, varying the position of the primordia in the bud, according to the different species; stamens with unilateral development and carpel with adaxial curvature. However, for Arapatiella and Jaqueshuberia it requires studies of this type to better understand the floral evolution of this diverse group. Here we also corroborate the discussion of the floral symmetry of the genus with data related to the zygomorphic and early development of the sepals and petals.

In this sense, with the present study, we corroborate the circumscription of the genus Tachigali based on shared ontogenetic characters. Future studies in conjunction with a more robust phylogeny of the genus allowed further analysis of the intraspecific relationships of the group.

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