1. Ornamentation patterns generated by mechanical perspectives
In this study, by comparisoning the FEM results of stress-driven buckling pattern on spheroidal core/shell structure and the ornamentation of spores and pollen in nature, clear and regular relationships between the ornamentation types and the two core parameters (shape factor and thickness coefficient) of spores and pollen were found, and most of the ornamentation in nature can be simulated by FEM (Figs. 2, 3 and 4). Our results indicated that stress is an significant factor to regulate ornamentation of spores and pollen, and the ratio of equatorial/polar radii and the ratio of effective size/exine thickness are two major factors that primarily drive the variation in the ornamentation (Figs. 2, 3 and 4).
Ornamentation patterns of fungi cover all seven stress-driven buckling pattern types (Clémençon, 1970, 1977; Pegler & Young, 1971, 1981, 1987; Fineran, 1994; Co-David et al., 2009; Clémençon, et al., 2012; Halbwachs & Bässler, 2015). For example, Rugate-smooth (type A; Fig. 2A), Smooth (type B; Fig. 2B) and Reticulate on poles - Circinate on equator (type C; Fig. 2C) are extremely rare in spores and pollen of plants but there are plenty of perfect instances in fungi (Elíades et al., 2006; Balajee et al., 2007). Although there are some complex types of ornamentation in fungal spores, the exine of most fungal spores appears to be thin and easily deformed (Co-David et al., 2009). The stress is the driving force in the formation of fungal spore ornamentation, which is resulted from the expansion mismatch between the exine and intine of spore wall.
In addition to that, several other factors can affect the performance of buckling on spheroids as well, mainly including the ratio of substrate/film elastic moduli and the ratio of maximum stress/critical stress (stress overload level of the film). However, these two factors have insignificant effect on the shape and surface morphology of the overall structure, mainly affecting the density and depth of wrinkles (Yin et al., 2008). Although these two factors have little influence, those four factors including ratio of equatorial/polar radii and the ratio of effective size/exine thickness can be freely combined to form a variety of fungal spore types. Moreover, there are also some spores may lack the exine to form ornamentation, such as the spores of Conocybe apala (a fungal species, HKAS69767) which appear extremely smooth (Fig. 3A3).
In conclusion, this study demonstrated the dependence of the stress-driven buckling patterns on the ratio of equatorial/polar radii and the ratio of effective size/exine thickness, reproduced the spore ornamentation patterns in nature, and provided a general explanation for the diversity of them (Figs. 2, 3 and 4). Actually, most wrinkles were idealized in the simulation results, but in real fungal spores, their ornamentation sometimes forms irregular shape, which may be affected by material unevenness and boundary constraints imposed by apiculus and plage etc.
2. Ornamentation patterns modulated by biophysical process and gene regulation
The plant spore and pollen walls have complex structures (McClymont & Larson, 1964; Olesen & Mogensen, 1978; Hesse 2000; Moran et al., 2007; Coelho & Esteves, 2011; Wallace et al., 2011; Denk & Tekleva, 2014; Wortley et al., 2015; Xu et al., 2016; Yang et al., 2020; Ma et al., 2021; Vaganov et al., 2021). Their formation processes are also complex, which generally include three stages: the development of the callose wall, the wavy morphology of primexine and the biosynthesis and transportation of sporopollenin (Wallace et al., 2011; Xu et al., 2016; Ma et al., 2021). Previous studies showed that the second stage - the wavy morphology of primexine - which is through a biophysical process called “phase separation”, is the key stage that determines the basic morphology of the ornamentation of plant spores and pollen (Heslop-Harrison, 1968; Radja et al. 2019; Liu et al. 2020). It has also been demonstrated that some proteins can avoid the formation of exine in some certain areas of the pollen surface, resulting in the development of germination pores, and thus determining the morphology of ornamentation (Radja et al., 2019; Liu et al., 2020; Zhang et al., 2020). These results suggest that biophysical process (such as “phase separation”) and gene regulation may act in combination to the formation of ornamentation in plant spores and pollen.
The main polymeric component of the exine of plant spore and pollen walls is sporopollenin. The property of it is strong and resistant to corrosion (Scott et al., 2004). In addition, the SEM and TEM images show that the exine of pollen wall is formed by extremely complex structures such as bacula, tectum and tryphine (Wortley et al., 2015). Therefore, our core/shell model may not be able to fully accommodate these kinds of complex structure, so further exploration and development of complex model are necessary in the later stage. Nevertheless, many types of them are also consistent with our simulation results, which means that although their structures are complex, they mainly consist of a two-layer structure. Therefore, similar results are eventually developed, indicating that stress is involved in the formation of pollen ornamentation structure.
However, by examining the SEM and TEM images of the plant spore wall ornamentation structures, it is evident that their structures are much simpler than those of the pollen with the exine structures clearly resembling wrinkles (Moran et al., 2007; Coelho & Esteves, 2011; Denk & Tekleva, 2014; Vaganov et al., 2021). Therefore, plant spores may have relatively simple structure due to their thinner exines, and stress-driven buckling may affect their ornamentation configurations to a certain extent.
It comes down to the fact that genetic factors may determine the shape and the exine thickness of spores and pollen (Wallace et al., 2011), leading in specific distribution of stresses on spore exine or pollen exine of different species, finally resulting in specific buckling patterns. With this study, “ornamentation” has been redefined. It mainly includes two types: “structure” and “pattern”. The ornamentation controlled by gene regulation is called “structure”, and the ornamentation through spontaneous buckling is called “pattern”. Ornamentation is the general term of the “structure” and “pattern”, but the formation mechanisms are vastly diverse.
3. Ornamentation patterns reformed from “secondary structure process”
The elastic deformation is the basic process of ornamentation formation, but secondary structure process produces more changes for the elastic-originated basic state. In this study, a “secondary structure process” in sporulation of some fungi species was observed under SEM, such as the spores of Strobilomyces mirandus (Fig. 3G1). Their ornamentation firstly bloom in stress-driven buckling with wrinkles forming on the spore surface. Then many upright ridges are produced along the wrinkles, which determine the final appearance of the ornamentation. This phenomenon provides idea for understanding other kinds of complex ornamentation which includes the hair-like ornamentation in Geastrum, the tufted ornamentation in Pisolithus, the rod-like to wing-like ornamentation in Auriscalpium and the verrucose ornamentation in Russula (Kuhar et al., 2012; Li et al., 2013; Lebel et al., 2018; Wang & Yang, 2019; Akyuz Yilmaz et al., 2021). Moreover, both the spores of Aspergillus rugulosus and A. spinulosporus correspond to the simulation results at R/t=100, k=0.5 (Fig. 3C1 and C4). They share a similar shape with two remarkable equatorial rings. However, the polar ornamentation of A. spinulosporus (Fig. 3C4) are warty rather than reticulate. This phenomenon may imply the occurrence of secondary structuring. Generally, more further studies are required to reveal the formation mechanism of these kinds of complex ornamentation.
In addition to the property of spores and pollen themselves, some environmental factors may also influence their ornamentation, especially those with thick exine. Higher thickness means more space for transformation (greater conversion space) driven by other external forces. When the exine is affected by external interference, such as dryness and erosion, the ornamentation of inelastic origin like fragmentation (debris), digestion and recombination may easily occur. For example, the intact exine of spore wall of Juncorrhiza casparyana (Fig. 3B1-1) is very thick, whose surface is flat and almost smooth, corresponding to the simulation result at R/t=5, k=0.9 (Fig. 3B1-1). However, there are also some spores produced by this species which have thinner exine and show many local uplifts, possibly from secondary structuring of inelastic origin (Fig. 3B1-2).
4. Ecological significance of ornamentation patterns
Spore and pollen ornamentation may directly or indirectly contribute to the fitness of fungi in the native environment (Kumaresan et al., 2012). This study indicated that a variety of ornamentation types can be produced only by changing the ratio of equatorial/polar radii and the ratio of effective size/exine thickness, which not only explored the evolution of ornamentation types, but also explored the influence of ratio of equatorial/polar radii and the ratio of effective size/exine thickness on ornamentation types from a more in-depth perspective. These fundamental works provided a theoretical basis for exploring the role of spore and pollen ornamentation in the process of ecological environment adaptation.
For instance, fungi specialize in various environmental conditions by changing shape factor and exine thickness. The spores of Aspergillus spp. that can infect human body are similar to red blood cells. They are oblate ellipsoids with abundant ring-like and reticulate patterns (Guarro et al., 2002; Balajee et al., 2007), which may increase the friction between them and body fluids, hinder sinking and improve suspension stability. Additionally, the spores of gastroid fungi and truffles that rely on animals to disperse spores often produce striate, reticulate or warty patterns (Læssøe & Hansen, 2007; Lebel & Catcheside, 2009), which may increase their retention time in animals to spread to farther habitats.