Dancing on the platform: Lability of floral organs of Beilschmiedia appendiculata (Lauraceae)

Abstract Floral characters are important for the systematics of the Lauraceae. However, structure and development of the flowers remain poorly known in the family. In this study, we observed the variation and early development of flowers of Beilschmiedia appendiculata, which belongs to the Cryptocarya clade of the family. The results indicate that the shoot apical meristems (SAMs) of the floral buds are enlarged and become a platform for the programmed initiation of the floral organs; floral organs develop basically in an acropetal pattern; phyllotaxis is whorled, initiation of floral primordia within a whorl is asynchronous; floral merosity is extremely variable, for example, dimerous, trimerous, tetramerous, dimerous plus trimerous, and trimerous plus tetramerous. In addition, this species has lost the innermost staminal whorl and glands are not closely associated with stamens of the third staminal whorl, which is unusual in the family Lauraceae. Our new observations broaden our knowledge of the variation of floral structure in Beilschmiedia and pose a fundamental question regarding the ecology underlying the lability of floral organs in B. appendiculata.

Floral merosity and organ number have frequently been used in generic delimitation of the Beilschmiedia group. Brassiodendron C.K. Allen has trimerous flowers possessing 6, two-locular stamens and was considered to be a heterogeneous group with some species ascribed to Beilschmiedia, while other species should be classified under Endiandra (Hyland, 1989;van der Werff, 2001).
Triadodaphne has trimerous flowers with 3 two-locular stamens and strongly unequal tepals and was either treated as a synonym of Endiandra or as a separate genus (Hyland, 1989;van der Werff, 2001). Both Sinopora and Hexapora have trimerous flowers with six stamens (Li et al., 2008;van der Werff, 2001). Species of the genus Syndiclis have both dimerous and trimerous flowers possessing variable numbers of stamens ranging from 4 to 9 (Zeng et al., 2017).
Allen including only L. appendiculata C.K. Allen and indicated that this genus is similar to Beilschmiedia but differs from the latter by the trimerous flower having six stamens. Lee and Wei (1982)  In this study, we investigated the early development and anatomy of flowers of B. appendiculata using both light microscopy and scanning electron microscopy to reveal morphological and structural variation of floral organs in the species and discuss its implications within a phylogenetic context.  Early in the initiation of the flower buds, the shoot apical meristem (SAM) was enlarged due to rapid peripheral growth, the SAMs were usually nearly round or fusiform. Basically, the flowers developed in an acropetal manner, that is, primordia of the outermost whorl first, followed by the inner whorls developed centripetally.

| MATERIAL S AND ME THODS
The three or four spaced tepal primordia of the outer whorl (P 1 ) were initiated first at the edge of the SAMs, but their initiations were asynchronous ( Figure 2a). Soon, the tepal primordia of the second whorl (P 2 ) started to develop between the primordia of the first whorl (P 1 ). The primordia of the second whorl were also asynchronous. Sometimes, a primordium of the first whorl was initiated somewhat later than the primordium of the second whorl, leading to a more prominent primordium of the second whorl than the primordium of the first whorl ( Figure 2a). Normally, the enlarged primordia of the first whorl were slightly broader than the primordia of the second whorl. In a normal flower, the primordia of the first whorl were equal in number to the primordia of the second whorl, and the six tepal primordia were decussate (Figure 2b,c). However, sometimes there were two primordia instead of one primordium ( Figure 2d).
The primordia of the third whorl (S 1 ) were initiated in line with the primordia of the first whorl (P 1 ) and alternate to the primordia of the second whorl (P 2 ) ( Figure 2d). For the lateral flowers of a cyme, the adaxial primordia were usually initiated earlier than the abaxial ones, which led to the adaxial primordia being more prominent than the abaxial primordia ( Figure 2d). The primordia of the fourth whorl (S 2 ) parallel to the primordia of the second whorl (P 2 ) were alternate to the primordia of the third whorl (S 1 ) ( Figure 2e). Timing of the primordial initiation of the fourth whorl (S 2 ) appears to be related to the parallel primordium of the second whorl (P 2 ) in the same row; that is, if one primordium of the second whorl (P 2 ) was initiated earlier, then the corresponding primordium of the fourth whorl (S 2 ) was also initiated earlier ( Figure 2e). Normally, the primordia of the first and the third whorls were equal in number, but sometimes, there were two primordia of the third whorl corresponding to one primordium of the first whorl ( Figure 2f). The primordia of the third and the fourth whorls developed further into fertile stamens. The primordia of the fifth whorl (S 3 ), corresponding to those of the third whorl (S 1 ), were alternate to those of the fourth whorl (S 2 ) (Figure 2g,h); these primordia normally develop into staminodes. There were only three staminal whorls in B. appendiculata, and the fourth staminal whorl is never initiated.
In a normal flower bud, the pistil consists of a single carpel (C) Normally, this species has flowers possessing two whorls of tepals, three staminal whorls, and one central pistil. The outer two whorls of stamens (S 1 and S 2 ) were introrse and fertile; the inner third whorl (S 3 ) was latrorse and usually sterile, but occasionally fertile ( Figure 2j). The fourth whorl of staminodes normally occurring in most Lauraceae was absent in this species.
The number of tepals, stamens, glands, and position of the glands varied greatly in the species. The number of tepals and stamens varied from 6 to 8, rarely 4 or 5 (Figure 3a,b). We counted 126 flowers and found that trimerous flowers are the most frequent merosity (54.8%, Table 1). The number of glands was usually variable, and these glands were often irregularly inserted on the disk of the nearby fertile stamens. Occasionally, the pistil possessed two carpels which were oppo- that is, the plastochrons and divergence angles change dramatically between whorls but are relatively equal or nearly so within a whorl.
The flowers of B. appendiculata are seemingly whorled, but floral organs are initiated asynchronously within a whorl (Figure 2a,b,d). For a lateral flower, the adaxial primordium is usually initiated earlier and FIGURE 2 Legend on next page is more prominent than the abaxial primordium ( Figure 2d). This developmental pattern seems common in the Lauraceae Kasapligil, 1951;Singh & Singh, 1985;Zeng et al., 2017). The floral phyllotaxis of Endiandra montana appears to be spiral (Hyland, 1989). Further studies are necessary in additional taxa of the family to better understand the details. The Lauraceae are primitive angiosperms, which may account for the asynchronous initiation of floral organs in a whorl.

| Tepals and androtepals
Tepals are sterile foliar organs surrounding the inner fertile elements, and there are usually six tepals arranged in two whorls in the Lauraceae (Rohwer, 1993). Different opinions exist regarding the origin of tepals. Buzgo et al. (2007) suggested that the tepals in the Lauraceae are derived from the abortion of stamens. Cronquist (1988) believed that the tepals in archaic angiosperms are modified leaves. Eames (1961) contended that the perianth had a dual origin in angiosperms, the inner whorl (i.e., petals) being derived from sterilization of stamens while the outer whorl (i.e., sepals) was modified from leaves.
According to our observations in this study, the tepals appear homologous to stamens. They are initiated in a similar series as foliar appendages on a condensed floral shoot, and there are gradual transitions between tepals and stamens, sometimes chimeras bear features of stamens and tepals in the position of tepals. In addition, the first staminal whorl is transformed into a third whorl of tepals in Dicypellium and Phyllostemonodaphne, and the two outer staminal whorls are tepaloid (Rohwer, 1993). Tepals are sometimes also transformed into stamens or completely reduced in Litsea and Lindera (Rohwer, 1993). Hyland (1989) reported that tepals developed into anthers or vice versa in Endiandra montana. In this study,

| Staminodes
Typical flowers of the family Lauraceae normally possess four different kinds of floral organs in a flower, that is, tepals in two whorls, fertile stamens in three whorls, staminodes in one whorl, and a central unicarpellate pistil. Our observations support the concept that the staminodes in the Lauraceae are reduced stamens with the major portion of the stamen reduced followed by fusion of lateral glands . In B. appendiculata, the staminode whorl in a normal Lauraceous flower has been lost and fertile stamens of the third staminal whorl are frequently found to be replaced by F I G U R E 2 Development of flowers of Beilschmiedia appendiculata. (a) Early development of a trimerous flower, the three primordia of the outer tepal whorl (P 1 ) with different prominence, arrow showing the position of a developing tepal primordium whose initiation is later than the tepal primordia of the inner tepal whorl; (b) a trimerous flower bud with initiation of all three tepals of the outer whorl, beginning of development of the inner tepal whorl, but the tepal primordia having different developmental rates, arrow showing the position of an expanding tepal primordium; (c) a trimerous flower bud with two whorls of well initiated tepal primordia; (d) a metamorphic flower bud with initiation of three staminal primordia (S 1 ), one additional tepal primordium (P) occurring near a tepal of the inner whorl, the adaxial primordium more prominent than the abaxial primordium; (e) a tetramerous flower bud, the four primordia of the first whorl of stamens (S 1 ) initiated, two upper tepal primordia more prominent than the lower two tepal primordia (P 2 ), the staminal primordia corresponding to the larger tepal primordia in the same row bigger than the staminal primordia corresponding to the smaller tepal primordia; (f) a trimerous flower bud, the left-side tepal primordium of the inner tepal whorl less prominent, two additional staminal primordia (S) occurring near two staminal primordia (S 1 ), both axillary to the outer whorl of tepals (P 1 ); (g) a regular trimerous flower, primordia of the third whorl of staminodes initiated, carpel primordium (C) also initiated; (h) a regular trimerous flower bud, glands initiated, arrow pointing to concavity of the apicallateral side of the carpel primordium; (i) the carpel primordium, arrow marking the position of a forthcoming ovule in the cross-zone; (j) a regular trimerous flower bud, arrow indicating the ovular protuberance, the two flanks of the carpel converging on one another from apical portion downwards to the cross-zone, the three primordia of the third staminal whorl having very different developmental rates; (k) the carpel with almost closed ventral suture; (l) further fusion of the ventral suture, the upper portion of the carpel elongated into style, the filaments prolonged; (m) a longitudinal section of the ovary displaying the two integuments of the anatropous ovule. Scale bars: a-c, and i: bar = 100 µm; e-h, j, and m: bar = 200 µm; k: bar = 300 µm; l: bar = 500 µm. ad, adaxial; C, carpel; G, glands; P, tepals of irregular occurrence; P 1 , the outer whorl of tepals; P 2 , the inner whorl of tepals; S, staminal organs with irregular position; S 1 , the first/outermost whorl of stamens; S 2 , the second whorl of stamens; S 3 , the third staminal whorl including either fertile stamens or staminodes; S 4 , the fourth staminal whorl including staminodes staminodes. Intermediate morphology between glands and stamens is frequently found in the third whorl of fertile stamens (Figure 3g,h), and epidermal cells of the glands and the secretory portion of the staminodes show similar morphological changes, both are granular before maturity. Sometimes fertile stamens of the third whorl become staminodes in B. appendiculata. This phenomenon was also observed in Syndiclis (Zeng et al., 2017). It seems that fertility of the innermost staminal organs increases the diversity of flowers.

F I G U R E 3 Metamorphic development in Beilschmiedia appendiculata. (a) A tetramerous flower bud
showing the more prominent adaxial primordium; (b) a dimerous flower bud, arrow pointing to two fused anthers of a stamen in the second staminal whorl (S 2 ); (c) two carpels face-to-face on the ventral side, each carpel bearing an ovule (O); (d) two opposite carpels connate at the base; (e) a young carpel bearing two ovular protuberances, the ventral suture down to the base of the carpel; (f) a carpel bearing two ovular protuberances at the cross-zone; (g) a fertile stamen fused to a lateral gland (G), arrow indicating an anther cell; (h) a fertile stamen (S 3 ) fused to a gland (G); (i) glands with irregular position and size, arrows pointing to the anther cells of a chimera of tepal and stamen; (j) a chimera of stamen and tepal, similar to a stamen; (k) a chimera of stamen and tepal, similar to a tepal, arrow indicating the anther cell; (l) a chimera of stamen and bract from the basal portion of an inflorescence, arrow showing the flap of a well-developed anther cell. Scale bars: a and c: bar = 100 µm; d-f, j and l: bar = 200 µm; b and k: bar = 300 µm; g and i: bar = 500 µm; h: bar = 1 mm. ad, adaxial; C, carpel; G, glands; O, ovule; P 1 , the outer whorl of tepals; P 2 , the inner whorl of tepals; S, staminal organs with irregular position; S 1 , the first/outermost whorl of stamens; S 2 , the second whorl of stamens; S 3 , the third staminal whorl including either fertile stamens or staminodes; S 4 , the fourth staminal whorl including staminodes However, it remains difficult to understand the underlying genetic network that regulates the development of the staminodes using only the fading borders model of Chanderbali et al. (2009). Probably the amount of expression of the B function plays a role in regulating the formation of staminodes.

F I G U R E 4
Illustrations displaying variation of floral organs of Beilschmiedia appendiculata. (a) A trimerous flower displaying the regular Lauraceous floral structure; (b) a dimerous flower displaying the regular arrangement of floral organs and glands, the third whorl of stamens becoming staminodes; (c) a mosaic flower of tri-and di-merosity displaying the fertile stamens of the third whorl, glands irregularly arranged, mosaics of stamens and glands, and mosaics of tepals and stamens; (d) a trimerous flower displaying the stamens of the third staminal whorl becoming staminodes or missing, glands irregularly arranged, two stamens sometimes in place of one stamen; (e) a mosaic flower of tetra-and tri-merosity displaying the stamens of the third staminal whorl becoming staminodes or missing, glands sometimes absent; (f) a tetramerous flower displaying the stamens of the third staminal whorl becoming staminodes or missing, glands sometimes absent, the pistil consisting of two carpels. Green crescents refer to tepals; gray solid circles indicate staminodes, yellow solid circles are fertile stamens, orange circles are glands, and red circles are pistils. P 1 , the outer whorl of tepals; P 2 , the inner whorl of tepals; S 1 , the first/outermost whorl of stamens; S 2 , the second whorl of stamens; S 3 , the third staminal whorl including either fertile stamens or staminodes; S 4 , the fourth staminal whorl including staminodes Li, 1982;Reece, 1939;Singh & Singh, 1985), thus pseudomonomerous in the family. Pseudomonomery is defined as a gynoecium consisting of seemingly only one carpel but actually representing several carpels merged and partially reduced. Vascular anatomy has been considered to be conservative and provides indications of vestiges of reduced or fused organs. The ovary in the Lauraceae possesses six vascular bundles, which was taken as evidence that the ovary in the Lauraceae was derived by fusion of multiple carpels, which was rejected by Rohwer (1993).
According to our observations in this study, the ovary of B. appendiculata normally consists of only a single carpel which is comparable to the free carpels in other primitive angiosperms, but rarely two free or basally fused carpels replace the unicarpellate pistil. Bi-or tri-carpellate status was also observed in Cassytha, Phoebe, Sassafras, Syndiclis, and Umbellularia (Feng, 1963;Kasapligil, 1951;Wang, 1995;Zeng et al., 2017). Previous authors hypothesized that the unicarpellate ovary, which is normal in the Lauraceae, was probably derived by reduction of a pluricarpellate pistil (Cronquist, 1981;Wang et al., 2000). It is well known that the gynoecium possesses one carpel per flower in the Hernandiaceae and Lauraceae, but is pluricarpellate in other families of the Laurales (Staedler, 2008). The unicarpellate gynoecium of Lauraceae is probably derived, because the basal family in the Laurales is pluricarpellate (Massoni et al., 2014;Renner, 2011).

| Number and position of ovules in the pistil
The pistil of the Lauraceae has been described as containing a single anatropous ovule (e.g., Kasapligil, 1951;Rohwer, 1993;Zeng et al., 2017). In this study, we observed a certain variability. There are sometimes two ovular primordia in the ascidiate carpel in B. appendiculata (Figure 3e,f). Within the Laurales, a carpel having two ovules is only found in the Calycanthaceae (Endress & Igersheim, 1997;Renner, 2004). The family Calycanthaceae represents the basalmost branch within the Laurales (Massoni et al., 2014;Renner, 2004). As a result, we consider that the two-ovuled carpel in B. appendiculata is a kind of reversal to the ancestral state. The one-ovuled carpel is a result of reduction in the two ovule states in the common ancestor of the core Laurales. Abortion of one of the two ovules was observed in the Calycanthaceae (Endress & Igersheim, 1997).

| Unusual floral characters of B. appendiculata
Beilschmiedia appendiculata was first described as a separate genus Lauromerrillia based on the flower having six stamens (Allen, 1942). Lee and Wei (1982) noticed that it has a variable number of fertile stamens: six or occasionally eight stamens, and thus incorporated Lauromerrillia into Beilschmiedia. We confirmed the observations of these earlier authors but provide more details. Beilschmiedia appendiculata does possess unusual flowers that are either trimerous or occasionally tetramerous, and the outer two staminal whorls are fertile whereas the third staminal whorl is sometimes partially fertile resulting in the variation of stamen number of the species. A similar situation also occurs in Syndiclis aff. malipoensis (Zeng et al., 2017).
However, our detailed study shows that the flower of B. appendiculata lacks the fourth staminal whorl which is usually present in other species as staminodes. Floral organs are reduced in miniature flowers of the Beilschmiedia group, but it is unusual to have reduced floral organs in a relatively large-flowered species as in B. appendiculata.
Despite the unusual features, B. appendiculata clearly belongs to the core Beilschmiedia group according to a recent phylogeny .
Reduction of stamens is parallel in the Beilschmiedia group and derived multiple times in the Beilschmiedia group because the sixstamened species are distributed in several clades according to recent phylogenetic studies Rohwer et al., 2014;Zeng et al., 2017). In China, there are two species of Beilschmiedia having six-stamened flowers. Besides B. appendiculata, B. pauciflora H.W.
Li from southern Yunnan also possesses flowers with six fertile stamens (Lee & Wei, 1982). The 2 six-stamened species together with the nine-stamened B. delicata and B. tsangii constitute a clade which receives low to moderate support (Li, 2020). Due to the lack of a robust phylogeny, it remains unclear whether the reduction of stamens of the two Chinese species resulted from a common ancestor or arose independently.
The presence and modification of glands have increased the diversity of flowers of the Lauraceae. In a typical flower of the family, each stamen of the third whorl normally possesses two glands at the base of the filament. In Brassiodendron, Chlorocardium, Phyllostemonodaphne, Urbanodendron, all fertile stamens are found to bear glands at the base of the filaments (Rohwer, 1993). In Pleurothyrium, the glands are distinctly enlarged surrounding the base of all stamens (Rohwer, 1993). In some species of Endiandra, the glands are concentrated into a glandular cushion (Hyland, 1989).
In Anaueria, Hexapora, and Williamodendron and a few species of Endiandra, Licaria, and Mezilaurus, the glands are completely lacking (Rohwer, 1993). The Chinese Syndiclis species were considered to have glands associated with stamens of the first whorl (Li, 1982).
However, a recent developmental study has shown that the glands are actually associated with stamens of the third whorl (Zeng et al., 2017). In B. appendiculata, the number and position of the glands were found to be variable (Figure 4c-f), the glands are irregularly inserted on the disk of the nearby fertile stamens but not associated with the stamens of the third whorl, and sometimes, the glands are fused to fertile stamens of the third whorl, which is unusual in the Lauraceae.
Flowers are an important component of diversified reproductive systems, and floral morphological diversity of angiosperms is closely related to animal pollination (Campbell & Powers, 2015;Ramos & Schiestl, 2019). It is evident that pollinators have an intense influence on floral traits (Gómez et al., 2014;Rusman et al., 2019).
Differentiation of pollinator visitation has been found between quaternary and quinary flowers in Ruta graveolens (Tang & Ren, 2011).
As a result, it is reasonable to infer that the lability  (Rohwer, 2009). Finally, both stamens and tepals become contiguous distally and the flower appears closed (personal observation). In B.
appendiculata, the flowers usually have six stamens belonging to the outer two staminal whorls, whereas stamens of the third whorl are frequently/partially specialized as staminodes. It seems unnecessary to have both glands and staminodes functioning at different times.
These structural changes may be related to functional shifts of the reproductive system in B. appendiculata, but it is unclear how the floral organs cooperate in six-stamened species such as B. appendiculata.

ACK N OWLED G EM ENTS
We thank Yi-Hua Tong for his kind help on collecting floral materials of Beilschmiedia appendiculata. We are also grateful to Jens G. Rohwer and an anonymous reviewer for their valuable suggestions. This work was supported by the National Natural Science Foundation of China [31970205 and 31770211] and the Metasequoia funding of Nanjing Forestry University.

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data used in the study are included in this paper.