https://orcid.org/0000-0002-8058-7168
ABSTRACT
Papaveraceae are known for their often showy flowers and diverse morphologies. Pteridophyllum racemosum (Siebold & Zucc.) is the only member in the Pteridophylloideae within the Papaveraceae, and its phylogenetic position has long been controversial, and a comprehensive analysis of its floral morphology was lacking. Our study focuses on the floral morphogenesis of P. racemosum. Histological sections complemented with scanning electron microscopy allowed a detailed characterization of P. racemosum floral development including description of the vegetative morphology, showing (1) the fine structure of the unusual pinnate leaves; (2) the well-defined floral structure and disymmetry; (3) a series of landmarks in floral development stages, with emphasis on the development of the gynoecium. Interestingly, the P. racemosum floral architecture is similar to that of the proposed ancestral Papaveraceae flower supporting the phylogenetic placement of P. racemosum as an early branching lineage within the Papaveraceae. Our comprehensive overview adds to the colorful library of floral diversity in Papaveraceae, providing a solid base for comparative analyses.
GRAPHICAL ABSTRACT
Introduction
The order Ranunculales is sister to all other eudicots and includes > 4500 species distributed among seven families (APG Citation2016). Ranunculales emerged approx. 130 million years ago (Magallón et al. Citation2015; Peng et al. Citation2023), and they are characterized by a large diversity in life-history traits, growth habit, leaf shape, flower and fruit forms, etc. The most common figures of merism of the floral whorls range from two to five, the perianth can be absent (Eupteleaceae) or, when present, differentiated into petals and sepals (bipartite) or composed of a single type of organ (unipartite), either sepaloid or petaloid. Additionally, floral organs vary extensively in form, size, color, and floral symmetry is also diverse, all in relationship with pollination modes (RanOmics Group Citation2023). In Ranunculales, several specific traits are found that are poorly represented among classical core eudicot or monocot model species, such as changes in merism, changes between whorled vs spiral phyllotaxy, or formation of novel organ types. Other traits that are found in Ranunculales are also known from core eudicots or monocots, such as radial vs. bilateral symmetry, spur formation, and transition between sexual systems in closely related taxa (Jensen and Kadereit Citation1995; Soza et al. Citation2012). Such homoplasious characters provide the opportunity to study the conservation of genetic mechanisms involved in the origin of homologous vs. convergent traits (RanOmics Group Citation2023). However, studying the origin and evolution of traits on a morphological or molecular scale requires precise knowledge of traits in a broad range of species. Key positions for comparative analyses hold those taxa that are sister to major lineages as they are indispensable for ancestral trait inference.
The Papaveraceae (poppy) family is one of the monophyletic families of the Ranunculales and they comprise four subfamilies: Fumarioideae, Hypecoideae, Papaveroideae, and Pteridophylloideae ( for comparison of their floral architecture). Molecular clock analyses date the age of poppy family stem group at 112–139 MYA and they have most likely diversified during the Cretaceous Terrestrial Revolution (Peng et al. Citation2023). Papaveraceae split already around 120 MYA into the Papaveroideae and the lineage that led to Hypecoideae, Fumarioideae and Pteridophylloideae. Interestingly, the majority of poppy family genera are biogeographically strictly restricted (Peng et al. Citation2023).
Figure 1. Floral morphologies in the Papaveraceae. (a) Eschscholzia californica (California poppy, Papaveroideae). (b) Pteridophyllum racemosum (pteridophylloideae). (c) Hypecoum imberbe (sicklefruit hypecoum, hypecoideae). (d) Lamprocapnos spectabilis (bleeding heart, Fumarioideae); floral diagrams are below the pictures with sepals colored in green, floral bract in dark blue, petals in grey, stamens in black, and the gynoecium as oval in the center. C, D referred to Sauquet et al. (Citation2015) and Hidalgo and Gleissberg (Citation2010); photos C, D, copyright free from Naturalist.org, C, by Quentin Groom, D, by Askalotl.
The phylogenetic placement of the Pteridophylloideae has been controversial, such that they were placed as the sister subfamily to the remaining Papaveraceae in a phylogeny that combined data on chloroplast DNA and nuclear 26S ribosomal DNA (Hoot et al. Citation2015). However, recent research has provided a comprehensive phylogenetic framework for Papaveraceae using full plastid sequences of 10 Papaveraceae species complemented with six plastid loci from 106 taxa. In this analysis, the Pteridophylloideae are placed as the sister group to both, the Hypecoideae and Fumarioideae (Peng et al. Citation2023; RanOmics Group Citation2023).
Pteridophyllum racemosum (Siebold & Zucc.) is the only species in the Pteridophylloideae subfamily. It is an evergreen perennial plant herb endemic to the central and northern regions of the Japan mainland (Honshu island) where it grows in the understorey of mountainous and subalpine forests (Tani Citation2001; Endo et al. Citation2011). They survive the winters by maintaining one- and two-year old leaves and winter buds, which develop right after flowering season and are surrounded by thick, protective bracts to allow fast reproduction in a short vegetative season, in contrast to most perennial herbaceous perennials that overwinter with complete shedding of old leaves (Tani et al. Citation2003; Endo et al. Citation2011). Pteridophyllum racemosum is self-compatible and reproduces mainly by seeds.
Ranunculales are notorious for their synthesis of diverse secondary metabolites, with benzylisoquinoline alkaloids (BIAs) being the economically most important. BIAs were also identified in root and aerial tissue of P. racemosum, among them protopine, sanguinarine, dihydrosanguinarine, oxysanguinarine, chelerythrine, magnoflorine, α- and β-allocryptopine (Ikuta and Itokawa Citation1976).
Despite the stunning morphology of P. racemosum, with the unusual combination of fern-like leaves and bell-shaped flowers and its distinct phylogenetic position in the Papaveraceae, a comprehensive analysis of its floral morphology is lacking. Here, we introduce the habitus of P. racemosum, and provide a detailed description of its floral morphogenesis by combining histological sectioning and scanning electron microscopy.
Materials and methods
Pteridophyllum racemosum plants were purchased from Dix Export bv (Heemstede, Netherlands), “A Touch of Green” Garden Webshop (Netherlands), and Pépinière AOBA nursery (La Touche au Burgot, France), transferred to the greenhouse and to a shady spot at the Botanical Garden, Giessen, Germany. The unique identifier given by the Giessen Botanical Garden for all plants is an IPEN number: XX-0-GIESS-2019-J-756. Photos were taken using a Leica DFC450 camera (Leica, Wetzlar, Germany) and a Leica M165 stereo microscope (Leica, Wetzlar, Germany).
Pteridophyllum racemosum buds develop during winter below the soil surface. For sampling, the soil surface was gently ruffled with a brush to collect the inflorescences. Fixing, embedding, sectioning and staining for histological analyses was done as described in (Becker et al. Citation2005). Briefly, ten intact inflorescences were fixed in freshly prepared cold FAA (3.7% formaldehyde, 50% ethanol, 5% acetic acid), vacuum infiltration was followed by overnight fixation at 4 ℃ (~16 h) and then dehydration in a graded ethanol series. Then they were embedded in Paraplast Plus (Roth, Karlsruhe, Germany) and cut into 8 µm thick sections using a Leica 2125RTS microtome (Leica, Wetzlar, Germany). After dewaxing, the sections were stained with 1% safranin and 0.2% fast green and analysed using a Leica DM 5500 microscope equipped with the above-mentioned camera (Leica, Wetzlar, Germany). The size of all floral buds was measured by ImageJ (1.53e; Wayne Rasband and contributors National Institutes of Health, USA).
For SEM, four intact inflorescences were fixed for two hours in freshly prepared glutaraldehyde fixative (2% glutaraldehyde, 0.5% paraformaldehyde, 0.2 M phosphate buffer, pH = 7.2) for 2 h at RT. After dehydration in an ethanol series, the samples were incubated twice in acetone for 1 h. Finally, samples were critical-point dried and sputter-coated at 35 mA for 35 sec, and then scanned with a LEO 982 scanning electron microscope (Zeiss, Jena, Germany) at 3kV.
Results
Morphology of the vegetative and reproductive organs
Pteridophyllum racemosum is a perennial, evergreen herb, with leaves organized in a rosette ). The leaves appear hardy and they are pinnate, reminiscent of a fern frond. Each leaf consists of around 25–30 leaflet pairs and multicellular trichomes form on the adaxial surface of the leaflet, while stomata emerge from the abaxial surface ). Winter buds contain a young inflorescence composed of many flowers. They form at or close to the center of the vegetative rosette and they are covered by several layers of protective bracts (Endo et al. Citation2011). The main inflorescence axis produces spirally arranged, subtending bracts and their axillary buds will give rise to lateral, secondary inflorescences with very short axes. This secondary inflorescence meristem will terminate in a single flower that opens first and produces subtending bracts with axillary buds from which secondary flowers emerge. 1–2 cm long pedicels connect the flowers with the inflorescence axis. Thus, the inflorescence type of P. racemosum is a thyrse, with lateral monochasia or dichasia. Flowers are disymmetric with two minute sepals, two whorls of two morphologically similar petals each, four fertile stamens in a single whorl and a syncarpous gynoecium composed of two carpels. Nectaries are absent. The petals are white, slightly shiny and oval in shape. The two outer petals occupy the outer petal whorl and two inner petals occupy the inner whorl. The stamens consist of a short, transparent filament and long anthers with two thecae ). Towards the end of floral bud development, the stamens extend above the gynoecium. At anthesis, stigmatic protrusion appear at the gynoecium apex and the gynoecium extends above the stamens ).
Figure 2. Pteridophyllum racemosum vegetative and floral morphology. (a) habitus of a flowering P. racemosum plant. (b) Pinnate leaf with leaflet numbers indicated. (c) scanning electron micrograph of the abaxial leaf epidermis including stomata. (d) SEM of multicellular adaxial trichomes, white lines indicated cell walls. (e) side view of the apical part of the inflorescence. (f) top view of a flower at anthesis. (g) view into the flower at anthesis with one petal removed. One sepal was falsely colored in yellow for clarity. Size bars correspond to (a, f) = 1 cm; (c) = 50 µm; (d) = 200 µm; (g) = 1 mm.
Floral morphogenesis
Floral morphogenesis commences with the initiation of a secondary inflorescence meristem at the flank of the primary inflorescence meristem with older secondary inflorescences forming basally ). The secondary inflorescence produces a terminal floral meristem and a second (and rarely third) floral meristem at its flank, both connected by the same peduncle to the primary inflorescence stalk ). Floral initiation occurs already under ground, before the main inflorescence axis expands. The two floral buds connected by one peduncle develop successively and our observations are all based on the later formed flower. shows a floral bud at stage 1, surrounded by a subtending bract and with emerged sepal primordia that will eventually encircle the dome shaped floral meristem ( summarizes the staging of flower development). Stage 2 floral buds show elongating, inward curving sepals and emerging petal primordia that elongate. The sepals have not covered the bud yet ). In stage 3, sepal primordia cover the floral bud, petals continue to elongate, and stamen primordia emerge and expand in length and width, surrounding the gynoecium primordium that becomes dome-shaped during stage 3 ). In stage 4 floral buds, petals and stamens continue to expand longitudinally, but the sepals discontinue their expansion, causing the petals to cover the floral bud at the apex. The gynoecium primordium gives rise to the carpel walls ). Two ovules emanate inside the gynoecium which elongates rapidly in stage 5 ). In stage 6, the two carpels fuse postgenitally at their apices and sporogenous tissue forms in the anthers ). In stage 7, the style elongates, inner and outer integuments form at the ovules, and pollen development continues in the anthers. All floral organs elongate, except for the sepals ). The bud extends in width from around 180 µm to more than 870 µm and in length from 100 µm to 950 µm during stages 1 to 7.
Figure 3. Histological sections showing different stages of P. racemosum floral development. (a) inflorescence meristems at the primary inflorescence apex. (b) longitudinal section through an inflorescence, subfigure assembled from multiple photos. (c) details of secondary inflorescence organization. (d) floral bud at stage 1 with sepal primordia initiated. (e) floral bud at stage 2, with petal primordia initiated. (f) floral bud at stage 2, when petals elongate. (g) floral bud at stage 3, when stamen primordia initiate. (h) floral bud at late stage 3, when stamen elongate and the gynoecium primordium becomes dome-shaped. (i) floral bud at stage 4, when the carpel walls initiates. (j) floral bud at stage 5, when carpel walls elongate and ovules initiate. (k) floral bud at stage 6, when carpels are fused at the apex. Longitudinal section subfigure assembled from two photos. (l) floral bud at stage 7, when the style elongates, integuments initiate, and male sporogenous tissue develops into pollen. (m) cross-section of floral bud in stage 7. Abbreviations: cw, carpel wall; fi, filament; fm, floral meristem; gp, gynoecium primordium; ii, inner integument; oi, outer integument; ov, ovule; p, petal; pim, primary inflorescence meristem; pp, petal primordium; s, sepal; sb, subtending bract; sim, secondary inflorescence meristem; sp, sepal primordia; stp, stamen primordium; st, stamen; sty, style; spt, sporogenous tissue; tt, tapetum tissue. Size bars correspond to (a, c, d, i-m) = 200 μm; (b) = 1 mm; (e-h) = 100 μm.
Table 1. Floral developmental stages of Pteridophyllum racemosum.
Spatial organization of floral organs and gynoecium morphology
The concurrence of floral developmental stages and the spatial arrangement of floral buds is shown in , where all terminal buds fully covered by petals are accompanied by buds of early developmental stages, and protective bracts are present within the inflorescence (Supplemental Figure). The base of the floral meristem is located between the primary flower and the flower subtending bract ). Later, the subtending bract meristem emerges and initiates laterally from the floral meristem shows the secondary inflorescence meristem at inception). In stage 3, two sepals of equal size cover part of the bud. In opposing position to the sepals, the outer whorl petals are initiated and the inner whorl petals form adjacent to the sepals. The four stamen primordia occupy the gaps between the developing petals, and from the center of the bud the dome shaped gynoecium primordium has already emanated ). In stage 4, the outer petals elongate and cover the inner whorl petals, stamens and initiated gynoecium ). In late stage 6, the gynoecium initiates the style ). Following carpel fusion, the stigma shows a slit positioned parallel to the inner whorl petals ).
Figure 4. Scanning electron micrographs of P. racemosum floral development. (a) top view on an inflorescence with some buds removed. (b) floral bud at stage 1, encircled by a bract. (c) floral bud at stage 1, surrounded by a bract, with a subtending bract initiating. (d) floral bud at stage 3 with all floral organs initiated. (e) floral bud at late stage 4 with elongated outer petals. (f) floral bud at stage 6 with some petals and stamen removed to reveal the fused gynoecium. (g) Stigma and stamens grow up to the same height in late stage 6. (h) side view on the gynoecium with all other floral organs removed. The inset shows the stigmatic papillae at higher magnification. (i) side view on the ovary. (j) enlargement of the future dehiscence zone. (k) outer surface of the ovary wall. Abbreviations: b, bracts; dz, dehiscence zone; fm, floral meristem; gp, gynoecium primordia; ip, inner petal; o, ovary; op, outer petal; sb, subtending bract; sbm, subtending bract meristem; sti, stigma; sp, sepal primordia; s, sepal; st, stamen; sty, style. Scale bars (a) 1 mm; (b-d) 100 μm; (e,f,j) 200 μm; (g-i) 500 μm, enlarged fig in h: 50 μm; (k) 50 μm.
The gynoecium consists of a short and wide superior ovary harbouring two ovules, a style around twice the length of the ovary and a stigmatic region. The latter has a deep slit in its center with stigmatic protrusions extending into the cleft ). As in the vast majority of the Papaveraceae, the dry dehiscent capsule of P. racemosum is derived from the syncarpous gynoecium. The replum is positioned at the outer sides of the mature fruit ). Conspicuous cells are regularly space across the ovary wall that have cell walls that do not reach the plane of surrounding cell’s cell walls ).
In summary, we describe the floral architecture and morphogenesis of P. racemosum, showing a symmetrical arrangement of floral organs in five concentric whorls. Unlike in most eudicots, the floral bud is not covered by the sepals as they stop elongating early in bud development (as observed for Capnoides sempervirens, data not shown). However, their protective function is taken over by the outer whorl petals which cover the inner whorl petals, stamens and gynoecium. The sepals remain on the pedicel when petals and stamens have dehisced already. Further, P. racemosum flowers include only four stamens, and only two seeds develop in each capsule, and both are rather low numbers when compared to other members of the Ranunculales order.
Discussion
Our detailed morphological analysis of P. racemosum shows the development of a textbook-like flower, in that we observed little deviation from the canonical flower architecture described for model plants like Arabidopsis thaliana with four sepals, four petals, six stamens and a two-carpellate gynoecium. We observed only minor differences: (1) only two sepals emerge from the floral primordium that cease expansion at an early developmental stage and persist on the flower after abscission of the other floral organs, (2) the four petals are arranged in two whorls that increase in size continuously throughout flower development, (3) four stamens emerge from a single whorl and (4) only two ovules are formed in the gynoecium.
Pteridophyllum racemosum flower is highly similar to the Papaveraceae ancestral flower
Ancestral state reconstructions of floral traits are available for the Ranunculales (Carrive et al. Citation2020), and because the Papaveraceae are sister to the Lardizabalaceae, Berberidaceae, Menispermaceae, and Ranunculaceae, their floral morphology is especially meaningful when deriving ancestral states. Only the Eupteleaceae branch earlier from all other Ranunculales, but as this family includes only two species with possibly derived traits, they are generally omitted from analysis (Carrive et al. Citation2020). Thus, Papaveraceae ancestral floral traits are in many cases similar to the Ranunculales ancestral traits. The ancestral Papaveraceae flower was reconstructed as follows: whorled, dimerous, disymmetrical perianth with 5–10 organs and three or more whorls. The perianth whorls were unfused, differentiated with a constant number of organs, and the inner perianth organs were petaloid. The ancestral number of stamens was less than six and two unfused, unilocular carpels in the center. Spurs and nectaries are absent (Hoot et al. Citation2015; Carrive et al. Citation2020). Interestingly, the flower of P. racemosum matches with the reconstructed ancestral trait flower in most aspects. However, it deviates from the ancestral Ranunculales flower, which was supposedly actinomorphic, with at least 12 free carpels (Carrive et al. Citation2020) in that it is dissymmetric with no differentiation between the two planes and has only two but fused carpels.
However, as an exception to typical Papaveraceae, P. racemosum does not develop crystal bearing idioblasts or laticifers and does not incorporate calcium oxalate crystals in the inner epidermis of the outer ovule integument (Hoot et al. Citation2015), but it remains unclear if these traits are ancestral states of the Papaveraceae.
Changes to zygomorphy in the sister lineage occurs late in flower development
In the most recent Papaveraceae phylogeny, P. racemosum is sister to both, the Hypecoideae and the Fumarioideae (Peng et al. Citation2023), but substantial changes to the ancestral Papaveraceae floral state occurred only in the Fumarioideae (), such as strongly modified perianth parts, zygomorphy, dissymmetry, and the development of nectaries at the stamen base (Damerval et al. Citation2013; Carrive et al. Citation2020). Interestingly, many stages of flower development are similar between P. racemosum and the zygomorphic Fumarioideae species Capnoides sempervirens: initiation of all floral organs is disymmetric in both flowers, but six stamens develop, of which four are monothecal and two dithecal in C. sempervirens (Damerval et al. Citation2013). Zygomorphic characters are achieved by unequal growth, organ fusion of stamens, and addition of a nectary spur in C. sempervirens later in development (Damerval et al. Citation2013).
Hypecoideae flowers are also disymmetric and with two sepals that are not enclosing the bud, they have four petals arranged in two whorls, four stamens that are arranged differently in that they are opposite of the inner whorl petals () and they develop nectaries. However, they are more proliferous with 30–100 seeds per capsule.
In comparison with the Papaveroideae, P. racemosum shares more characters with the Hypecoideae and Fumarioideae, as the Papaveroideae have more than 16 stamens, the sepals generally enclose the buds, most species have a higher number of petals, and many have more than two carpels (Hoot et al. Citation2015). Both Hypecoum and Pteridophyllum are characterized by actinomorphic androecia of four dithecal stamens (Sauquet et al. Citation2015). Thus, the floral characters support the recent phylogeny of P. racemosum being sister to the Hypecoideae and Fumarioideae (Peng et al. Citation2023). However, the absence of laticifers is unique to P. racemosum (Hoot et al. Citation2015), but could be a character that was lost in this species.
The presented work was limited by the availability of plant material, which is rarely grown in botanical gardens throughout Europe and the lack of protocols for growing the specimens. Further, access to the buds is challenging as they slowly develop after the flowering season below the substrate surface and when the inflorescence axis elongates, most stages of flower development have already passed. Additional analyses on the timing of bud development and dehiscence of the capsules would be very interesting, but requires more plant material.
Conclusions and outlook
This work provides a comprehensive overview of P. racemosum floral morphogenesis and detailed description of the floral morphology, which is identical to the reconstructed ancestral Papaveraceae flower. The key position of P. racemosum in the Papaveraceae family as sister to the dissymmetric Hypecoideae and the Fumarioideae in which zygomorphy originated once or twice, contributes to its importance for comparative analysis in terms of floral morphogenesis, but also for specialized secondary metabolites.
While P. racemosum is difficult to grow under controlled greenhouse conditions without a harsh winter, it can be maintained in cold frames or Botanical gardens in temperate regions, allowing for more extensive studies regarding its physiology, specialized metabolism, and unusual leaf morphogenesis.
Author contributions
AB drafted and supervised this research. DK performed specimen photography, paraffin sectioning, and SEM. KE provided and optimized the SEM protocol and supervised SEM. DK and AB wrote the manuscript. All authors read and agreed to publish the final manuscript.
Supplemental Material
Download TIFF Image (29.2 MB)Acknowledgements
We thank Till Strohbusch for taking care of the plants. We are also grateful to Helena Pillich and Sabine Agel from the Imaging Unit at Justus-Liebig-Universität Gießen for helpful advice for the SEM work.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Supplementary data
Supplemental data for this article can be accessed online at https://doi.org/10.1080/23818107.2024.2352773
Data availability statement
Original data have been deposited here: http://dx.doi.org/10.22029/jlupub-18415.
Additional information
Funding
Notes on contributors
Doudou Kong
Mr. Doudou Kong is a graduate student of Biolog under the supervision of Prof. Dr. Annette Becker, whose primary are of interest is comparative expression analysis in Ranunculales.
Katrin Ehlers
Dr. Katrin Ehlers is a cell biologist, whose primary interest is in the development and evolution of plasmodesmata and diverse microscopic techniques.
Annette Becker
Prof. Dr. Annette Becker is a plant geneticist, whose primary interest is in the origin and evolution of flowering plants.
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