The land pyrenoid:

A Silurian way to deal with heat and light?

 

Richardson, D.A. – Cambridge University: Plant Science Dept.

Abstract

Early Silurian land plants (plants living 450 Million years ago), inferably similar to the living hornwort (my experimental plant), could have made use of a pre-existing pyrenoid CO₂ Concentrating Mechaism (CCM) - inherited from an algal ancestor - not because CO₂ was in short supply in the aerial environment; (7000ppm CO₂; 20 times greater than today's CO₂ conc.), but in response to higher temperature and more intense light conditions compared to the water column. The land CCM, in the Silurian atmosphere could have responded to high  Photosynthetically Active Radiation (PAR) as my experimental hornwort CCM’s did; by increasing efficiency for CO₂ in the photosyntheic process at the cost of a reduced light use efficiency; the CCM acting as - a ''light use efficiency reducer'' (based on results) - and at high temperatures the CCM up regulates, also increasing efficiency for CO₂, acting as - an ''oxygenase reaction reducer'' - (based on results). The hornwort CCM thus can be likened to a primitive stomata, regulating CO₂ uptake, in response to light, temperature and thallus water content variations. The eventual loss of the pyrenoid CCM, and the move towards a more advanced morphology, (as seen in the C₃ liverworts), meant CO₂ was no longer being pumped around the active site of Rubisco, thus early C₃ liverwort Rubisco kinetics would have improved efficiency, moreover, early C₃ liverworts evolved new chloroplast architecture to deal with high PAR in a more efficient way. Also, the evolution of pores, to reduce water loss, made the early C₃ liverworts less dependant on water to photosynthesise, than the desiccation prone solid thallus of the hornwort.

Bryophyte Phylogeny

Liverworts and hornworts are a successful group of non-flowering, rootless lower plants, grouped as bryophytes. They possess a polykiohydric nature, namely their water content tends to adjust to the moisture conditions of their environment (Deltoro et al 1998). This condition is markedly different from that of the tracheophytes, which are homohydric, where water supply (from roots) and water status are maintained by stomatal apparatus which prevent the desiccation of the photosynthetic tissue. Unusually though, an ancient living group of bryophytes named hornworts possess stomata in the sporophyte but not the gametophyte (REF).

Polykiohydry in land plants appears to be a much more ancient state than homoihydry, with Mega-fossil evidence of the early land plants shown to be liverwort-like in ultra structure (Niklas 1997; Edwards et al 1998). However within the range of surviving orders of bryophytes, progressive specialisation of their morphology show evidence of a movetowards greater control over water relations; from simplesolid thallus (Pellia spp.) to the more complex differentiated structures with pores (Marchantia spp.) that show more control over water status.

In terms of the origin and evolution of early land plants, modern day bryophytes quite possibly resemble the characteristics of the earliest plants on land. The first good evidence for the existence of bryophyte-like land plants (Eoembryophytes) is seen in spore tetrads (comprising four membrane-bound spores), found over a broad geographic area in the mid-Ordovician period, 476 Myrs (Gray 1993). The combination of decay resistant walls (implying the presence of sporopollenin) and tetrahedral configuration (implying haploid meiotic products) are further diagnostics of land plants. Further evidence lay in spore wall ultra structure and the structure of fossil cuticles from the Late Silurian and Devonian mega fossils, leading Kendrick and Crane (1997) to suggest the above palaeobotanical evidence would support previous arguments that land flora during these times was liverwort-like. 

According to Kendrick and Crane (1997), land plants (embrophytes) are most closely related to the Charophyceae, a small group of predominantly freshwater green algae. Within this group, either Coleochaetales (15 living species) or Charales (400 living species) or a group containing both, is a sister group to land plants. Land plant monophyly is supported by comparative morphology and gene sequences (18S cox iii).

Relationships among the major basal living groups are uncertain. But the best supported hypothesis resolves liverworts as basal and either mosses or hornworts as the living sister group to vascular plants (tracheophytes).

From water to air

The transition from an aqueous medium, in which the ancestral Charophyceae group lived, to a gaseous medium, exposed the early land plants to new physical conditions. 

For instance, in place of the structural support of unlimited water the first land plants in an aerial environment faced desiccation exposure and the compressive effects of gravity. The early thalli would have also been exposed to relatively higher photon flux density, which would previously have been exponentially attenuated by a water column, and a 10⁴ gain in diffusion rate of CO2 as water places such diffusive limits (Osmund et al 1982). 

Consequently, key physiological and structural adaptations, over time, needed to occur in early land plants. Development of cutins to reduce water loss probably evolved from pre-existing elements of the primary metabolism in the ancestral charophycean algae (REF). Additionally, photoprotective mechanisms possibly developed to cope with higher photon fluxes in aerial environments utilizing aspects of the already existing photo respiratory pathways (Kendrick and Crane 1997). However it is unknown whether the photoprotective role of photorespiration evolved in photosynthetic organisms in shallow water at high light intensity or in the early land plants.

Rubisco

The effect of the land invasion by early land plants probably had a profound effect on the carboxylating enzyme ribulose 1,5 bisphosphate carboxylase / oxygenase, Rubisco, a photosynthetic enzyme, that had evolved in an aquatic media for some 3,800 million years into an aerial CO2 concentration during the late Ordovician early Silurian of approx. 5400 – 7000ppm CO₂ (Berner 1998), without the diffusive barriers placed on CO2 by water.

Rubisco catalyses the first step of the dark reaction side of the photosynthetic reaction termed the Calvin cycle (REF). The first step of this reaction involves Rubisco covalently attaching CO₂ to a 5 carbon sugar, RuBP, and the simultaneous hydrolysis of the six carbon intermediate to form 2 molecules of PGA, of which one bears the carbon introduced from CO₂.

However, Rubisco has a poor ability to distinguish between CO₂ from the O₂ molecule, perhaps because there is no formal binding site for CO₂. Consequently, when RuBP is bound to an active site of Rubisco, it can be attacked by O₂, producing the two products; 2 – phosphoglycolate (P-glycolate) and PGA, a process termed an oxygenase reaction. To salvage lost C as a result of oxygenation of RuBP, the photorespiratory cycle (evolved earlier) recycles P-glycolate to release C at an energetic cost (REF). 

The poor kinetic properties of Rubisco, along with diffusive limitation to the passage of CO₂ through water and cell boundary layers were all limiting factors to photosynthesis in the aquatic medium placing a strong selection pressure towards the development of mechanisms for increasing CO₂ concentrating around the Rubisco molecule, mechanisms termed, Carbon Concentrating Mechanisms (CCMs), possibly evolving 3400 MYA. The effect of raising the internal CO₂ environment using CCMs (sometimes by 50 – 100 fold would and does counteract the above constraints). Whether the early land plants retained the CCM is uncertain, however, an inference may be made to suggest a pyrenoid-based CCM was still present in some, if not all, early land plants (before c3 orientation), as the pyrenoid (only found in microalage) is found also in a group of byrophytes from the Class Anthocerotae.

The biophysical CCM

The most extensively studied Biophysical CO₂ Concentrating Mechanisms CCM's have been in cyanobacteria which use a carboxsome-based CCM, small polyhedral-shaped protein bodies containing both Rubisco and the enzyme Carbonic anhydrase (CA) which catalyses the dehydration of HCO3- to CO₂ (Badger & Andrews 1987; Bowes 1993).

CA

H⁺ + HCO₃^-  CO₂ + H₂O

In micro algae and some hornworts, an equivalent structure to the carboxysome is the pyrenoid, a starch coated protinaceous structure present in the chloroplast, believed to play a similar role (Badger et al 1993). to microalage, with a pyrenoid-based CCM (Pronina & Semenko 1992; Badger 1998). 

 

To summarize the makeup of an generalized CCM, 4 components are needed; (1) a mechanism whereby rapid interconversion between CO₂ and HCO₃- can take place, extracellularly and intracellularly, the latter occurring at typical stromal pH values within the Rubisco-containing compartment or more effectively, at low pH in the thylakoid lumen; (2) a Ci-transport mechanism at plasma membrane, chloroplast envelope or both; (3) ATP energy to power Ci transport; (4) a diffusion barrier to prevent CO₂ from diffusing away from Rubisco (Smith & Griffiths 2000). 

It has been established that the first two features of the above list have been shown to involve CA in a range of eukaryotic algae and cyanobacteria which utilise CCMs and it has recently been suggested that CA might function as a diffusion barrier (Raven 1997).The pyrenoid structure thought to be associated with the CCM found in many micro algae (Badger et al 1994; Amoroso et al 1998; Woods 1999) and lichenised algae (Palmqvist 1993), has also been observed in some species of byrophytes from the class Anthocerotae (REF). 

Moreover, Vaughn (1992) established, using immuno-gold labelling, that the enzyme Rubisco was found within the pyrenoid structure. 

Smith & Griffiths (1996b), further established that Anthoceros crispus and Phaeoceros laevis (Smith & Griffths 2000) exhibited low compensation points, low K_0.5 and a CO₂ uptake and release pool after photosynthesis had been inhibited, using the C-reduction cycle inhibitor, glycoladehyde, (a method term light-dark transients), revealing a CO₂ pumping mechanism termed a Dissolved Inorganic Carbon pump (DIC pump). 

All of the above have been used as physiological CCM diagnostics in cyanobacteria and micro algae. To date, Phaeoceros laevis and Anthoceros crispus have been shown to possess an operative CCM showing similar characteristics to micro algal CCMs. But why retain this ancient microalgal relic in the aerial environment? Considering approx 450-500 million years ago CO₂ Concentrations were (5400 –7000ppm) or indeed independently evolve the same (or similar) mechanism, at a latter period, during a drop in atmospheric CO2?

With or without a CCM

It has been observed that not all species from the Anthocerotae class possess a pyrenoid state (Vaughn 1990). For example all genus of the multiplastidic Megaceros spp. and two uniplastidic species, Anthoceros fusiformis and Phaeoceros coriaceus, do not possess a pyrenoid-based CCM. 

This is further borne out by carbon isotope studies where the delta values for Megaceros moandreus and Megaceros endivifolis (herbarium samples) show C3 responses. Whereas on-line measurements on Anthoceros crispus and Anthoceros agrestis (sporophyte) showed a C4-like response, although there is no evidence C4 or CAM biochemistry (pers com). 

This situation raises some intriguing questions as to the evolutionary significance of the "no pyrenoid condition" in uniplastidic cells of some hornworts and liverworts that have assumed a C3 orientation.

Hornwort CCM variability

Andrews Hanson and Badger (Functional Plant Biology Volume 29 Number 2 & 3 2002) examined hornwort CCM function by using a combined fluorometer/mass spectrometer based technique to compare pyrenoid-containing (Phaeoceros Prosk. and Notothylas Sull.) and pyrenoid-lacking (Megaceros Campbell) hornworts, with the liverwort Marchantia polymorphaL. that has standard C₃ photosynthesis and a thalloid growth form similar to hornworts. They found that Notothylas has more CCM activity than Phaeoceros, and that Megaceros has the least CCM activity. Notothylas and Phaeoceros had compensation points from 11–13 parts per million (ppm) CO₂, lower K_0.5(CO₂) than Marchantia, with negligible photorespiration, and they accumulate a pool of dissolved inorganic carbon (DIC) between 19–108 nmol mg^–1 chlorophyll. Megaceros had an intermediate compensation point of 31 ppm CO₂ (compared with 64 ppm CO₂ in Marchantia), a lower K_0.5 (CO₂) than Marchantia, and some photorespiration, but no DIC pool. They also determined the catalytic rate of carboxylation per active site of Rubisco for all four species (Marchantia, 2.6 s^–1; Megaceros, 3.3 s^–1; Phaeoceros, 4.2 s^–1; Notothylas 4.3 s^-1), and found that Rubisco content was 3% of soluble protein for pyrenoid-containing species, 4% for Megaceros and 8% for Marchantia.

Diffusive limitations

Physiological reasons for the retention of a CCM on land, or indeed its recent evolution in response to falling atmospheric CO₂, is still unclear, as the diffusive limitations of water are no longer limiting and unlike algae they are not moving up and down a water column, facing sudden shifts in HCO₃- and light. However, diffusive limitations might have still been acting on the early liverwort-like plants due to heavily cuticlised solid thalli (prior to air chamber evolution), to reduce water loss. Furthermore without pores or stomata (as seen in modern day C3 liverworts) the conductance of CO₂ within the solid thallus might have been low enough to require the retention of a CCM. 

 

Robe, Richardson & Griffiths, (unpublished) conducted a detailed comparative study to evaluate the relative costs and benefits of a biophysical CCM in Phaeoceros laevis (one chloroplast per cell with an unventilated thalli) as opposed to photosynthesis being based on the diffusive supply of CO₂ in a number of liverworts ranging from Pellia spp. representing the unventilated thalloid structure similar to Phaeoceros, together with liverworts showing increasing levels of complexity and ventilation (viz Conocephalum, Marchantia and Lunularia). The results showed that the limitations to gas exchange in an unventilated thallus such as Pellia, were so great as to render minimal rates of CO₂ assimilation, with a high internal conductance to CO₂ (gi); meanwhile, the advantages of the CCM in Phaeoceros were to restore the rates of CO2 assimilation and electron transport to those equivalent to the highly ventilated bryophyte thallus of Lunularia. 

In conclusion, the operation of the CCM certainly does ensure that most of the Anthocerotae can compare with other more” advanced” bryophytes.

Land pyrenoid CCM and Low Nitrogen

Apart from a possible advantage a CCM might provide a solid pore-less thallus over CO₂ conductance, (Beardall et al. 1982; Raven et al 1985) suggest that the CCM may confer another advantage, namely improved nitrogen use efficiency of growth. This is hypothesised to result from the idea that less nitrogen has to be diverted to the synthesis of Rubsico, and possibly photrespiratory enzymes. In an early Silurian aerial environment, nitrogen availability and acquisition may have been limiting compared to a water column as the case may still be today in certain environments (Crittenden, Katucka & Oliver 1994). As the Rubisco molecule has a low turnover rate then retention of a CCM that conveys Nitrogen efficiency would be advantageous (Raven and Lucus 1985). However, this has not been conclusively demonstrated.

Land pyrenoid CCM - reducing oxygenation

Another advantage of a CCM in a land plant such as the Anthocerotae would be to reduce the oxygenase reaction in the enzyme Rubisco by elevating the CO₂ Concentration around its active site, reducing competition from oxygen. The unique chloroplast architecture of the more “advanced” Anthocerotae posses thylakoids that cross the pyrenoid, termed channel thylakoids, Burr (1970), which have been speculated (through inference from work with algae) by Makay & Gibbs (1991) to be dominant in photosystem I and not Photosystem II (water splitting side of the light reaction, releasing oxygen), therefore reducing further oxygenation events.

Land pyrenoid CCM and high light

Growth in high light conditions might be another environmental factor acting to retain or effect the operation of a CCM on land. This factor has been noted in a study by Smith et al (1998) whom investigated a hypothesis that cyanobiont lichens (with CCM) growing under contrasting microhabitats show inter-specific and intra-specific variation in photosynthetic responses, which could be correlated with the variations in the degree of expression of the biophysical CCM. It was found that populations of Peltigera membranaecea, from exposed crags showed more pronounced CCM activity (by the accumulation of a larger Ci pool) than populations from shaded deciduous oak woodland. 

A possible explanation for these observations were that an active CCM will effectively reduce the light-utilization efficiency of photosynthesis (Palmqvist et al 1994c), therefore, increased CCM activity as a strategy of optimising the supply of CO₂ to Rubisco, might be most profitable in environments where CO₂ is a more limiting resource than light, i.e exposed habitats where lichens are subject to high PAR. 

An investigation into the way liverworts (with increasing morphological specialisation, including the hornwort Phaeoecros laevis) deal with excess of light has so far not been in studied. Moreover how the Anthocerotae CCM responses to increased light intensity needs to be evaluated.

Land pyrenoid CCM and desiccation

The retention of pyrenoid CCM on land may be useful in the response of these polykiohydric organisms to variations in environmental stresses, such as high light, but also more importantly at fluctuating thallus water contents. Smith et al 1998, conducted a investigation on the effect of the uptake and release pools of CO₂ in the lichen Peltigera membranaecea at varying thallus water contents using a method termed light dark transients. It can be inferred from the data that a 10 fold drop in CO₂ up take and release pool sizes occur when thallus water contents decrease from optimal (5.1- 6.2 mg g-1 d.wt) to (2.3 mg g-1 d.wt) a 63% reduction in water content.

Additionally, calculations by Green & Snelger (1982), show that when Monoclea spp. (a liverwort with a solid thallus, with no air pores) is compared to Marchantia spp.(with water proof cuticle penetrated with air pores), maximum photosynthesis is only slightly greater in Marchantia, but air spaces giving greater advantage over Monoclea for water relations. However, the solid thallus of Monclea showed superior photosynthetic ability in very moist environments, possibly like the hornwort (with CCM).

The pyrenoid CCM and high/low CO2

Finally, fluctuations in external CO2 concentrations have been a popular line of investigation in marine and freshwater algae with CCMs (Matsuda, Bozzo and Colman 1998; Woods 1999; Colman 2000) 

It has been recently demonstrated that in cells of Chlamydomonas reinhardii, external CO₂ concentrations can affect the active uptake of CO₂ and HCO₃- and has been show to be suppressed in Chlamydomonas reinhardii grown in high external CO₂ after about 8 days in 5% CO₂ (pers com Colman 2000). After this period the new generation of cells lost the CCM capacity. However when transferred to a low CO₂ external environment, active transport of CO₂ and HCO₃- within 2hrs of acclimation, and CA ext activity increased 10 fold after 6 hours after acclimation to 0.035% Co2. It has been proposed that the CCM is induced when the CO₂ concentration in the medium is reduced to a critical level. 

Matsuda, Bozzo and Colman (1998) have suggested the possibility of a CO₂ sensor at the green algal cell surface, which under high CO₂ growth conditions would cause repression of the CCM, whereas under CO₂ depletion the sensing mechanism would initiate a signaling cascade culminating in the derepression of the CCM. Woods (1999) also was able to down regulate the CCMs in Chlorella spp. and Trebuxia spp .with a 2 day 5% CO₂ treatment and then upregulated it with low CO2 treatment only after 2 hours.

Carbonic anhydrase and the land pyrenoid

The possibility of being able to “switch off” or down regulate the operation of the Anthoceroate CCM is not unrealistic in the light of recent work by Smith and Griffiths (2000) who established that CA plays a role in the operation of the CCM in Phaeoceros laevis , as in all microalage investigated. Smith and Griffiths (2000) conducted an investigation into the role of CA in photosynthesis and the activity of the CCM in Anthocerotae by using the membrane-permeable CA inhibitor ethoxzolamide (EZ), drawing a comparison to a range of liverworts and mosses. 

The results showed that inhibition of assimilation occurred in all bryophyte treated with EZ, however the degree of inhibition was greatest in Phaeoceros laevis. Furthermore, there was a pronounced decline in Ci-uptake efficiency and a decrease in the initial slope of CO2-affinity curve at low external levels where Ci-uptake efficiency in other liverworts were unaffected. There were no significant differences between the convexities of the light response curves in Phaeoceros which would indicate a diversion of ATP to energise the CCM. 

In studies on light dark transients on Phaeoceros treated with EZ a speculation made was that although active transport of CO₂ was still occurring (due to the appearance of a CO2 release pool), the Ci transported to the stroma is not being utilized by Rubisco when CA is suppressed by EZ. This point may raise the question as what the exact mechanism of Ci transport is if EZ does not suppress the active uptake of CO₂ in the Anthocerotae. 

The major drawback with using EZ to manipulate the operation of the Anthocerotae CCM in future studies is that CA is involved in other non CCM photosynthetic processes, as shown by the depressions of gross assimilation rates in mosses of 65 % and 50% in other non CCM based liverworts treated with EZ. 

 

The usefulness of being able to "switch of " or down regulate the Phaeoceros CCM with a high CO2 environment will be critical in elucidating the operation of the Anthocerotae CCM. However in terms of the ecology of Phaeoceros, a relatively rapid response (of approx. 14 days) to variations in external CO₂ (initially 5% CO2) may not be seen, due to the fact that fluctuations in CO2 in the aerial environment is relatively minimal, or at least not as irritate as in a water column.

Discussion of results

The following investigation revealed that when the gametophyte of extant hornworts is exposed to external CO₂ concentrations of either 5%, ambient or 0.00175%, (for 14 days), the CCM showed no effect in its operation, unlike algal CCM’s where 5% CO₂ after only 2-8 days switches the CCM off (REF). This indicates that a hornwort tissue response, to differing external CO2 concentrations, has not occurred, (maybe more time is needed in these CO₂ environments). However, in hornworts grown at 30⁰C for 14 days (compared to those at grown at lower temperatures) there was a sig. diff. in the operation of the CCM, appearing to increase the capacity of dissolved inorganic carbon (DIC) uptake and lower the CO₂ compensation point and, lower K _0.5 CO₂, (with hydrated thallus). Therefore an advantage of a CCM in a land plant such as the Anthocerotae would be to reduce the oxygenase reaction in the enzyme Rubisco by elevating the CO2 Concentration around its active site, reducing competition from oxygen. 

 

The unique chloroplast architecture of the more “advanced” Anthocerotae possess thylakoids that cross the pyrenoid, termed channel thylakoids, Burr (1970), which have been speculated (through inference from work with algae), by Makay & Gibbs (1991), to be dominant in PSI, not PSII (water splitting side of the light reaction, releasing oxygen), therefore reducing further oxygenation events, possibly making up for a Rubisco with low specificity. 

 

Furthermore, high light intensity (compared to low light intensity grown plants), can have a sig. effect on the capacity of the hornwort CCM; with a large dissolved inorganic carbon (DIC) uptake and low CO₂ compensation point and low K _0.5 CO₂. An active CCM will effectively reduce the light-utilization efficiency of photosynthesis, therefore increased CCM activity will optimise the supply of CO₂ to Rubisco helping it deal with high PAR. The hornwort appeared to be able to photosynthesise at Photon Flux Densities (PFD) above 1500umols m². s², (in those grown at 120umol m². s² for 14 days); However a constituently expressed CCM at high PAR kept NPQ low, causing PSII damage, maybe acting as an electron sink to power the DIC pump, and/or as a proton sink in the thylakiod lumen, suplying protons to the reaction; (H⁺ )+ HCO₃^- CA CO₂ + H₂O, so lowering protonation of the xanthophylls reducing NPQ. However, at an extremely low light treatment, Pmax was greater than that of high light treated plants (lower Pmax could have been caused by chlorophyll loss), indicting growth at low PFD is preferred in the hornwort. 

 

However light adaptability is possible with use of the CCM’s plasticity as a ''light use efficiency reducer'' a high PAR and an ''oxygenase reaction reducer'' at high temperature. Early land plants in the Silurian atmosphere, 450MYA, may well have made use of the already existing pyrenoid CCM (as possessed by the ancestral Coleochales), not because external CO₂ was in short supply (Silurian aerial environment; [CO₂] 5400 - 7000ppm) (REF), but as a way to deal with high PAR by increasing efficiency for CO₂ at the cost of a reduced light use efficiency, the CCM acting as - a ''light use efficiency reducer'' - and in high temperatures the CCM up regulates, also increasing efficiency for CO₂ acting as - a ''oxygenase reaction reducer''. 

 

The hornwort CCM can be likened to a primitive stomata, regulating CO₂ uptake, in response to light, temperature and thallus water content variations. The eventual loss of the pyrenoid CCM, and the move towards a more advanced morphology as seen in the C3 liverworts, meant a change in shape of the chloroplast from band shaped to discoid (REF). The discoid shape would allow more light adaptability; an example of this is the unusual light dependant changes in the chloroplast morphology in species of hornworts without pyrenoids (Burr 1968). Brown a lemon (19--) suggests the evolution of the multiplastic cell (away from the uniplastic cell found in Phaeoceros) may have meant the end of pyrenoid containing plastids. In the mutiplastidic cell the division of a number of plastids would have been more difficult to co-ordinate. An even distribution of Rubisco in the stroma would not require a pyrenoid division to insure that Rubisco is present in every chloroplast. With Rubisco no longer being pumped CO2 around the active site, the early C3 liverwort Rubisco kinetics would have improved efficiency, coupled with morphological specialisation to aid CO₂ influx, and pores to reduce water loss, thus making the early C3 liverworts less dependant on water to aid CO₂ in flux.

To retain a pyrenoid CCM on land meant adaptability to the Silurian atmosphere; higher temperatures and stronger light conditions (REF) than experienced in the water column. To retain a pyrenoid CCM on land today means hornworts with CCM’s have the plasticity to adapt to high and low temperatures and very low light, to higher light environments, ranging from the Australian and India rain forests, to, ditches in Scotland and Canada. However, full hydration of the thallus is essential for efficient CCM activity, so a damp to wet environment is the habitat where they are found. If environmental conditions are no longer favourable, hornwort tuber formation can occur and the thallus dies (REF); the plant to be resurrected when environmental conditions are suitable. This is another useful adaptation to cope with the high PAR and drying summer conditions experienced by Phaeoceros growing in the Mediterranean area.