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Funaria hygrometrica: (A) population of gametophytes with attached and expanded sporophytes; (B) a mature sporophyte topped by a calyptra; (C) immature, spear-shaped sporophyte covered by a calyptra-the calyptra is divided into a narrow, apical rostrum and a swollen inflated base that narrows in diameter at the bottom, and the sporophyte apex extends into the upper most portion of the rostrum (arrow), during early developmental stages. Scale bars: (A) ¼ 4 mm; (B) ¼ 2 mm; (C) ¼ 1 mm.
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The maternal gametophytic calyptra is critical for moss sporophyte development and ultimately sporogenesis. The calyptra has been predicted to protect the sporophyte apex, including the undifferentiated sporogenous region and seta meristem, from desiccation. We investigate the hypothesis that this waterproofing ability is due to a waxy cuticle. The...
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... moss Funaria hygrometrica Hedw. is a cosmopolitan, pioneer species found in disturbed habitats. Sporophytes range from 12 to 80 mm tall (Fig. 1A, B; Miller and Miller, 2007) with structurally complex capsules that can contain more than 100 000 wind-dispersed spores (Shaw, 1991). The calyptra of F. hygrometrica is 2 -5 mm long, including a narrow rostrum above a wider inflated base (Fig. 1C). Plants used in the study were from a Connecticut population (CONN Budke #145) cul- tured ...
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... a cosmopolitan, pioneer species found in disturbed habitats. Sporophytes range from 12 to 80 mm tall (Fig. 1A, B; Miller and Miller, 2007) with structurally complex capsules that can contain more than 100 000 wind-dispersed spores (Shaw, 1991). The calyptra of F. hygrometrica is 2 -5 mm long, including a narrow rostrum above a wider inflated base (Fig. 1C). Plants used in the study were from a Connecticut population (CONN Budke #145) cul- tured in the laboratory in PlantCon plant tissue culture containers (MP Biomedicals, Solon, OH, USA) on a rich sandy loam soil mix. Gametophytes were grown from spores of the original population for at least 4 months at room temperature under 16 h of ...
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... were flooded with water for 24 h and remained in the cold growth chamber for 1 week after un-flooding. They were then placed at room temp- erature under 16 h of daylight. Calyptra and sporophytes from this laboratory population were harvested at a late stage of spor- ophyte development (capsule expanded with calyptra present on the apex; Fig. 1B) and used for all subsequent experiments. Additionally, mature gametophytes with fully expanded leaves were collected from this ...
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... addition to gross morphological differences, the rostrum and inflated base of the calyptra (Fig. 1C) differ in the thick- ness of their cuticles ( Fig. 5A-F; cuticle thickness indicated by arrows minus the space indicated by an asterisk). These two regions differ significantly in the thickness of several indi- vidual cuticle layers and the number of cells in the circumfer- ence (Table 1). The narrow rostrum had a thicker cuticle, ...
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... bars: (A) ¼ 20 mm; (B) ¼ 500 nm. 1975b), is completely nested inside the rostrum (Fig. 1C). At this developmental stage, the inflated base exclusively covers mature portions of the seta below. The thicker cuticle on the rostrum may be functioning to prevent desiccation of these zones that are integral to spore and seta formation. In studies of vascular plants, cuticle thickness and waterproofing ability are uncorre- lated ( ...
Citations
... The stereome is outlined by a cuticle, common to both, seta and stem. The presence of a cuticle in bryophytes has been known for a long time, as pointed out by Strunk (1914), Hébant (1977) or, more recently, by Koch et al. (2009), Budke et al. (2011), Busta et al. (2016 or Glime (2017c). Here, we follow Jeffree (2006) and recognize a bi-layered structure with thin CP and CWP, as observed by Sack and Paolillo (1983) in Funaria hygrometrica, while Budke et al. (2011) pointed it out as a multi-layered structure. ...
... The presence of a cuticle in bryophytes has been known for a long time, as pointed out by Strunk (1914), Hébant (1977) or, more recently, by Koch et al. (2009), Budke et al. (2011), Busta et al. (2016 or Glime (2017c). Here, we follow Jeffree (2006) and recognize a bi-layered structure with thin CP and CWP, as observed by Sack and Paolillo (1983) in Funaria hygrometrica, while Budke et al. (2011) pointed it out as a multi-layered structure. The histochemical analyses with Sudan B on cross sections (seta and stem) confirm its lipidic composition (Table 2). ...
... Besides, it has been long suggested (Proctor 1979) that n-alkanes, considered as part of the cuticular wax in vascular plants, could have a similar role in bryophytes. The n-alkanes of bryophyte origin have been used as biomarkers in paleoenvironmental reconstructions in mires (see, for instance, ortiz et al. 2011, 2016), although they could contribute to slow down water loss (Budke et al. 2011(Budke et al. , 2013. Meanwhile, their true role in mosses remains to be determined. ...
... The gametophyte composes the vegetative and, by definition, the reproductive body: it represents the free-living generation that produces gametes, undergoes sexual reproduction, and supports the resulting zygote and sporophyte. In some mosses at least, the gametophyte also controls sporophyte development via the calyptra, which covers the sporophyte apex (Budke & al., 2011Budke & Goffinet, 2016;Budke, 2019). The sporophyte is permanently attached, physiologically dependent on the maternal plant, and hence shorter-lived, and functionally limited to producing and dispersing spores to complete the cycle. ...
Traits of the spore‐bearing generation have historically provided the basis for systematic concepts across the phylogenetic spectrum and depth of mosses. Whether taxa characterized by a simple sporophytic architecture are closely related or emerged from independent reduction is often ambiguous. Phylogenomic inferences in the Funariaceae, which hold the model taxon Physcomitrium patens , revealed that several such shifts in sporophyte complexity occurred, and mostly within the Entosthodon‐Physcomitrium complex. Here, we report the rediscovery of the monospecific, Himalayan endemic genera Brachymeniopsis and Clavitheca , after nearly 100 years and 40 years since their respective descriptions. The genera are characterized by, among other traits, their short sporophytes lacking the sporangial peristome teeth controlling spore dispersal. Phylogenomic inferences reveal that Brachymeniopsis gymnostoma arose within the clade of Entosthodon s.str., a genus with typically long‐exserted capsules. We therefore propose to transfer B. gymnostoma to the genus Entosthodon , as E. gymnostomus comb. nov . Furthermore, Clavitheca poeltii , the sole species of the genus, is morphologically highly similar to E. gymnostomus , and should also be transferred to Entosthodon , but is retained as a distinct taxon, E. poeltii comb. nov . , until additional populations allow for testing the robustness of the observed divergence in costa and seta length between the Nepalese and Chinese populations.
... In bryophytes, the presence of a cuticle has been revealed using histochemical and electron microscopy techniques in the gametophores and sporophytes of mosses, liverworts and hornworts (Schönherr & Ziegler, 1975;Moore et al., 1982;Schofield & Hébant, 1984;Cook & Graham, 1998;Buda et al., 2013;Busta et al., 2016;Li et al., 2018;Salminen et al., 2018;Budke, 2019;Resemann et al., 2019;Kong et al., 2020;Glime, 2021;Kriegshauser et al., 2021). Certain characteristics of tracheophyte cuticles, such as the presence of cuticular waxes and the differentiation between cuticle layer and cuticle proper, have also been identified in bryophytes (Budke et al., 2011;Buda et al., 2013;Busta et al., 2016;Matos et al., 2021a,b). The cuticle of the leafy gametophore of the model moss Physcomitrium patens is, indeed, structurally and chemically similar to that of tracheophytes, although with higher proportions of phenolic acids (Buda et al., 2013;Renault et al., 2017;Li et al., 2018). ...
Terrestrial mosses have been used for more than 50 years to monitor air pollution. We argue, however, that their value as biomonitors is based on two widespread but partially erroneous assumptions concerning their morphological structure (the structural myth) and physicochemical characteristics (the physicochemical myth). The structural myth consists of the oversimplification of the moss morphological structure. The physi-cochemical myth recognizes their high cation exchange capacity (CEC) as the only pathway for pollutant uptake. Here, we gather and discuss the evidence demonstrating that these assumptions are misleading and focus the discussion on the aspects that make mosses good biomonitors of air pollution. First, we show that these plants (i) do have a cuticle, whose structure and composition differs among species, (ii) can have epidermal cells, that differ in shape and thickness from other cell types, (iii) have a vascular system, whose degree of development is lineage-and species-specific, and (iv) have rhizoids, that can absorb water, nutrients and pollutants. The effect of these traits in the pollutant uptake processes has been understudied in biomonitoring studies. Second, we show that mosses (i) do not concentrate as much pollutants as they could according to their high CEC, (ii) can retain large amounts of particles containing atmospheric pollutants in their surfaces, and (iii) in many contexts, the spatial structure of the concentrations of pollutants in mosses depicts the pattern expected for atmospherically deposited particles. Thus, the quality of these organisms as biomonitors of air pollution also lies in their high capacity to retain particles. We do call for more research on how moss structural and physiological traits affect pollutant uptake dynamics and recommend researchers using mosses as air quality biomonitors to face critically to inaccurate or insufficiently demonstrated assumptions in this context.
... Despite recent efforts to demonstrate the properties of cuticles on the surface of both the gametophyte and sporophyte phases of bryophytes, most studies focus on Setaphyta (Proctor, 1979;Haas, 1982;Clayton-Greene et al., 1985;Neinhuis and Jetter, 1995;Koch et al., 2009;Xu et al., 2009;Budke et al., 2011;Budke et al., 2012;Buda et al., 2013;Busta et al., 2016;Matos et al., 2021a;Matos et al., 2021b) but not on hornworts. ...
... Histochemical and transmission electron microscopy (TEM) analysis revealed the presence of a thin cuticle covering the air pores of gametophyte thalli in Marchantia species (Schöenherr and Ziegler, 1975). This layer was also detected through a series of studies using TEM in both life cycle stages of the Funariaceae moss species, being more stratified in the sporophyte in comparison with the gametophyte (Budke et al., 2011;Budke et al., 2012;Buda et al., 2013;Budke and Goffinet, 2016). The main wax classes identified in the gametophytes of mosses and liverworts were fatty acids, primary alcohols, alkanes, and esters (Benesǒváet al., 1972;Heinrichs et al., 2000;Heinrichs and Rycroft, 2001;Xu et al., 2009;Matos et al., 2021a). ...
... A structure of a thin layer outermost the epidermis corresponding to the cuticle was also verified by TEM on the gametophytes of the liverwort Marchantia (Schöenherr and Ziegler, 1975) and of Funariaceae species (Budke et al., 2011;Buda et al., 2013). The more stratified layer in the sporophyte in comparison to that of the gametophyte found in the present study has also been found for mosses (Budke et al., 2011;Budke et al., 2012;Buda et al., 2013;Budke and Goffinet, 2016). ...
The development of a hydrophobic cuticle covering the epidermis was a crucial evolutionary novelty ensuring the establishment of land plants. However, there is little information about its structure and chemical composition, as well as its functional implications in avascular lineages such as Anthocerotophyta. The main goal of the present study was to compare the gametophyte and sporophyte cuticles of Phaeoceros laevis. Semithin sections were analyzed through light microscopy (LM), cuticle structure was evaluated by transmission electron microscopy (TEM) and epicuticular wax morphology was analyzed by scanning electron microscopy (SEM). Total waxes were analyzed by CG/MS, and the components were identified based on the mass spectra. A thin lipophilic layer was detected on the sporophyte surface, structured as a stratified cuticular layer, similar to the well-known structure described for vascular plants. On the other hand, the gametophyte cuticle was observed only with TEM as a thin osmiophilic layer. SEM analyses showed a film-type wax on the surface of both life phases. The wax layer was eight-fold thicker on the sporophyte (0.8 µg cm⁻²) than on gametophyte (0.1 µg cm⁻²). Possible mechanical and/or drought protection are discussed. Fatty acids, primary alcohols, and steroids were identified in both life phases, while the kauren-16-ene diterpene (3%) was detected only on the sporophyte. Although no alkanes were detected in P. laevis, our findings unveil great similarity of the sporophyte cuticle of this hornwort species with the general data described for vascular plants, reinforcing the conservative condition of this character and supporting the previous idea that the biosynthetic machinery involved in the synthesis of wax compounds is conserved since the ancestor of land plants.
... An additional interaction between the gametophyte and sporophyte phases occurs at the distal end of the sporophyte where the immature sporophyte apex is covered by the gametophyte Evansia 38 (3): 2021 101 calyptra (Budke 2019). The calyptra has a relatively thick cuticle (Budke et al. 2011) that is formed early in development and protects the sporophyte apex until capsule maturation (Budke et al. 2013). In contrast, the cuticle of the young moss sporophyte is relatively thin (Budke et al. 2012) and exposure to low humidity at this stage, without the presence of the calyptra, results in wilting of the apex and often sporophyte death (pers. ...
... The population was then placed back into the original room temperature growing conditions to facilitate sporophyte development. The methods described here are modified from Budke et al. (2011). ...
Moss sporophytes are physically attached to and dependent on the leafy gametophyte through their entire life. During early development, the sporophyte apex is covered by the calyptra, a cap of gametophytic tissue which protects the developing capsule. The aim of this study is to examine the influence of the calyptra on sporophyte transpiration rates in mosses. We used two laboratory-grown species with different sporophyte and calyptra sizes for this study: Funaria hygrometrica Hedw. and Physcomitrium pyriforme (Hedw.) Hampe. When the calyptra was removed from the sporophyte apex, there were significantly higher rates of sporophyte transpiration compared to individuals with the calyptra present. These results provide evidence supporting the importance and influence of the gametophyte calyptra on the movement of water in moss sporophytes. Changes in transpiration, and thus the pull of water and nutrients from the gametophyte into the sporophyte, may also have downstream effects on sporophyte reproductive output.
... 29,30 In addition, cuticular wax composition analysis revealed that Funaria hygrometrica, another drought-tolerant moss, has vascular plants-type cuticular wax enriched with wax esters, and different moss tissues such as leafy gametophyte, calyptra and sporophyte capsule surfaces have their distinct wax compositions, suggesting that cuticle biosynthesis is tightly regulated by environmental and developmental cues in mosses. 31 Indeed, P. patens cuticles have different compositions in gametophores and protonema stages. 30 In contrast with drought-tolerant mosses P. patens and F. hygrometrica, liverwort Marchantia polymorpha, and hornwort Anthoceros agrestis gametophytes have cuticles containing high levels of fatty acids and phenolic compounds such as hydroxybenzoic acid and coumaric acid. ...
As an adaptive innovation in plant terrestrialization, cuticle covers the plant surface and greatly contributes to the development and stress tolerance in land plants. Although past decades have seen great progress in understanding the molecular mechanism of cuticle biosynthesis in flowering plants with the contribution of cuticle biosynthesis mutants and advanced cuticle composition profiling techniques, origins and evolution of cuticle biosynthesis are poorly understood. Recent chemical, phylogenomic, and molecular genetic studies on cuticle biosynthesis in early-diverging extant land plant lineages, the bryophytes, shed novel light on the origins and evolution of plant cuticle biosynthesis. In this mini-review, we highlighted these recent advances in the molecular biology of cuticle biosynthesis in bryophytes, and provided evolutionary insights into plant cuticle biosynthesis.
... During the early stages of development, the unexpanded sporophyte was completely enclosed in a humid microenvironment provided by the calyptra (Figure 2B; Budke et al., 2011Budke et al., , 2012Hiss et al., 2017). To ascertain whether stomatal development is initiated at this early developmental stage, the calyptra of young sporophytes was removed and the underlying epidermis was checked for the presence or absence of stomatal lineage cells (Figures 4A,B). ...
Mosses are an ancient land plant lineage and are therefore important in studying the evolution of plant developmental processes. Here, we describe stomatal development in the model moss species Physcomitrium patens (previously known as Physcomitrella patens) over the duration of sporophyte development. We dissect the molecular mechanisms guiding cell division and fate and highlight how stomatal function might vary under different environmental conditions. In contrast to the asymmetric entry divisions described in Arabidopsis thaliana, moss protodermal cells can enter the stomatal lineage directly by expanding into an oval shaped guard mother cell (GMC). We observed that when two early stage P. patens GMCs form adjacently, a spacing division can occur, leading to separation of the GMCs by an intervening epidermal spacer cell. We investigated whether orthologs of Arabidopsis stomatal development regulators are required for this spacing division. Our results indicated that bHLH transcription factors PpSMF1 and PpSCRM1 are required for GMC formation. Moreover, the ligand and receptor components PpEPF1 and PpTMM are also required for orientating cell divisions and preventing single or clustered early GMCs from developing adjacent to one another. The identification of GMC spacing divisions in P. patens raises the possibility that the ability to space stomatal lineage cells could have evolved before mosses diverged from the ancestral lineage. This would have enabled plants to integrate stomatal development with sporophyte growth and could underpin the adoption of multiple bHLH transcription factors and EPF ligands to more precisely control stomatal patterning in later diverging plant lineages. We also observed that when P. patens sporophyte capsules mature in wet conditions, stomata are typically plugged whereas under drier conditions this is not the case; instead, mucilage drying leads to hollow sub-stomatal cavities. This appears to aid capsule drying and provides further evidence for early land plant stomata contributing to capsule rupture and spore release.
... 1C), whereas, hornworts lack a calyptra. The moss calyptra is a detached cap of tissue formed from the maternal gametophyte (Fig. 1D,E from Budke et al. 2011) and is present in some form on all of the approximately 13,000 species of mosses. ...
... Acid resistance in bryophytes has been attributed to phenolic compounds that can be present in either the cell wall or cuticle (Kroken et al. 1996;Kodner & Graham 2001), thus Janzen's observations of acid resistance cannot be definitively attributed to only a cuticle and may indicate that the cell walls and/or cuticle of the calyptra rostrum contain higher levels of phenolic compounds. Following these experiments, the advent of electron microscopy has enabled direct observation of thin bryophyte cuticles (Budke et al. 2011;Cook & Graham 1998;Koch & Frahm 2007;Proctor 1979;Sack & Paolillo 1983). Structural and developmental studies, using electron microscopy, have demonstrated that the calyptra cuticle of Funaria hygrometrica is significantly thicker than the leafy gametophyte cuticle (Budke et al. 2011) and the calyptra cuticle develops precociously relative to the sporophyte cuticle , with the potential to prevent dehydration early during sporophyte development. ...
... Following these experiments, the advent of electron microscopy has enabled direct observation of thin bryophyte cuticles (Budke et al. 2011;Cook & Graham 1998;Koch & Frahm 2007;Proctor 1979;Sack & Paolillo 1983). Structural and developmental studies, using electron microscopy, have demonstrated that the calyptra cuticle of Funaria hygrometrica is significantly thicker than the leafy gametophyte cuticle (Budke et al. 2011) and the calyptra cuticle develops precociously relative to the sporophyte cuticle , with the potential to prevent dehydration early during sporophyte development. Chemistry analyses have also determined that the cuticular waxes are thicker per unit area on the calyptra compared to the rest of the maternal gametophyte (Busta et al. 2016). ...
The calyptra is a small, maternal gametophyte structure that covers the apex of the offspring sporophyte and is critical for the transition from seta formation to capsule differentiation in many moss taxa. It has been hypothesized to function by (1) providing a mechanical constraint that coordinates the development of the presumptive capsule, (2) secreting hormones that influence sporangium differentiation, (3) physically protecting the undifferentiated capsule region, (4) preventing water loss from the sporophyte apex, and/or (5) lowering the rate of sporophyte transpiration. This review explores the experiments that have tested these hypotheses and the evidence that has been found to support or refute them from scientific literature dating back to the late 1700's. Across mosses, calyptrae come in a wide array of shapes and sizes, which may correlate with aspects of morphology, habitat, and function. The overarching objective of this paper is to renew discussion about and research into this small, but important moss organ. ©2019 by The American Bryological and Lichenological Society, Inc.
... The moss Funaria hygrometrica Hedw. is a cosmopolitan species characteristic of disturbed and urban habitats, distinguished from other North American species in the genus by a combination of traits to include a large, revoluble annulus, lanceolate endostome segments, and a strongly twisted and hygroscopic seta (Miller & Miller 2007). This species is amenable to laboratory culture and undergoes sexual reproduction when exposed to shorter daylengths and colder temperatures (Budke et al. 2011;Dietert 1980). Desiccation tolerance of the sporophyte is assisted by the cuticle of the maternal calyptra (Budke et al. 2013). ...
Desiccation tolerance (DT) in poikilohydric organisms is dependent upon four principal sequential factors, (1) the rate of drying (RoD), (2) the water content (WC) of the organism deriving from the equilibrating relative humidity (RHeq), (3) the duration of the desiccation event, and (4) the rate of rehydration (RoR). The first two factors are often combined in experiments as the “intensity” of desiccation, and thus the effect of one on the other is relatively unknown but likely to be important ecologically. We hypothesized that more protracted rates of drying should mitigate damage at lower equilibrating RHs in a known inducibly DT species. Cultured uniclonal shoots of the moss Funaria hygrometrica were dried at different RoDs (time from full turgor to leaf curling, from 0.067 to 120 h) at five different RHs (12, 33, 54, 75 and 93%), allowed to equilibrate at each RH, rehydrated and assessed using chlorophyll fluorescence at 0.5 and 24 h postrehydration. At 24 h postrehydration, shoots of F. hygrometrica subjected to a rapid-dry event (<10 min) were unable to recover to control levels of fluorescence (Fv/Fm and ΦPSII <80% of control levels) when equilibrated at RHeqs of ≤54%. Recovery was improved when the rate of drying was extended for more than ∼15 hours from full turgor to the onset of leaf curling at these RHeqs. When shoots were rapidly dried at RHeqs >54%, recovery was complete. At 0.5 h postrehydration, Fv/Fm levels indicated moderate damage at all RHeqs except 93%, with no mitigation of damage when the rate of drying was extended. Intensity of desiccation is normally construed as the ability to tolerate a combination of rate of drying × equilibrating RH. By varying the rate of drying across a range of RHeqs, F. hygrometrica is found to be able to tolerate all water contents tested, providing the rate of drying is extended to at least 15 h from full turgor to leaf curling. By adopting either the “step-down” approach to drying or the “wet substrate” technique used here, future studies on the desiccation tolerance of poikilohydric organisms can more accurately assess responses to desiccation stress.
... These cells form the sporophyte that ultimately consists of the foot (embedded in the maternal gametophyte and functioning in water/nutrient transfer), the seta/stalk, and the capsule ( Figures 5A,B). Throughout early development the sporophyte apex is topped by the calyptra, a maternal cap of gametophyte tissue, that is covered by a relatively thin cuticle that protects the immature sporophyte from water loss (Budke et al., 2011;. Moss sporophytes are initially green and capable of photosynthesis, but they do not fix sufficient carbon to sustain their entire growth and development (Proctor, 1977). ...
