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Anatomical, morphological and ecophysiological strategies in Placopsis pycnotheca (lichenized fungi, Ascomycota) allowing rapid colonization of recently deglaciated soils
Flora 206 (2011) 857–864
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Anatomical, morphological and ecophysiological strategies in Placopsis pycnotheca (lichenized fungi, Ascomycota) allowing rapid colonization of recently deglaciated soils Asunción de los Ríos a,∗ , José Raggio b , Sergio Pérez-Ortega a , Mercedes Vivas b , Ana Pintado b , T.G. Allan Green b,c , Carmen Ascaso a , Leopoldo G. Sancho b a
Museo Nacional de Ciencias Naturales, CSIC, C/Serrano 115 dpdo, E-28006 Madrid, Spain Departamento de Biología Vegetal II, Facultad de Farmacia, Universidad Complutense de Madrid, E-28040 Madrid, Spain c Biological Sciences, University of Waikato, Private Bag 3105, Hamilton, New Zealand b
a r t i c l e
i n f o
Article history: Received 11 January 2011 Accepted 20 March 2011 Keywords: Anatomy Cephalodia Gas exchange Receding glaciers Soil crust Terricolous lichen Nitrogen fixation
a b s t r a c t The green algal lichen Placopsis pycnotheca was identified at Pia and Marinelli glaciers (Isla Grande of Tierra de Fuego, Chile) as a primary colonizer of bare soil in areas close to the front of the glacier or around small ponds created after glacier retreatment. Electron microscopy study showed that P. pycnotheca formed a thick hypothallus within which hyphae and their extracellular polymeric substances bind numerous soil particles. This structure augments water holding and soil stabilization capacities and constitutes an early stage in soil crust development. In addition, numerous cephalodia are formed within the hypothallus and subsequently develop upwards towards the thallus surface, sometimes before the formation of squamules with green algae. These anatomical and morphological strategies together with physiological properties such as the long photosynthetic activity period (measured in the laboratory) help explain its pioneering role as a colonizer and its apparently high growth rate. © 2011 Elsevier GmbH. All rights reserved.
Introduction Ecological research analyzing the primary succession on newly exposed soil surfaces after deglaciation is central to advancing our understanding of ecosystem dynamics and for evaluating potential effects of climate warming. Primary succession is generally carried out by microorganisms and cryptogamic organisms that rapidly colonize deglaciated soils well in advance of the vascular plants (Burrows, 1990; Kastovska et al., 2005; Nemergut et al., 2007; Schmidt et al., 2008; Wynn-Williams, 1993). Cyanobacteria, green algae, lichenized and free living fungi, mosses, and heterotrophic bacteria are typically the first organisms to colonize the soil surface and subsurface altering the properties as they form a biological soil crust (Belnap et al., 2001; Breen and Levesqué, 2008; Evans and Johansen, 1999).
∗ Corresponding author. E-mail addresses: [email protected] (A. de los Ríos), [email protected] (J. Raggio), [email protected] (S. Pérez-Ortega), [email protected] (M. Vivas), [email protected] (A. Pintado), [email protected] (T.G.A. Green), [email protected] (C. Ascaso), [email protected] (L.G. Sancho). 0367-2530/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.flora.2011.05.002
The lichen genus Placopsis (Agyriaceae, lichenized Ascomycota) is a widespread early colonizer of rock surfaces and bare soils, such as recently deglaciated areas, especially in subantarctic regions, with 22 species currently known from southern South America (Follmann et al., 1991; Galloway, 2002, 2010). Placopsis species, although having a green algal primary photobiont, also have cyanobacteria in cephalodia and are efficient nitrogen fixers. They, and other cephalodial and cyanobacterial lichens, can contribute to the nitrogen economy in areas where this nutrient is limiting (Breen and Levesqué, 2008). Placopsis pycnotheca I. M. Lamb is known from southern Argentina, the Juan Fernández Archipelago (Chile), from latitude 32◦ S to 54◦ S in the Chilean Andes and from South Georgia (Galloway, 2002, 2010). It is characterized by its crustose terricolous thallus, composed of swollen areoles and squamules, encrusting sand, soil and small pebbles; and by the simple, subglobose to finger-like isidia. During our study of the colonization of recently deglaciated areas in Tierra de Fuego, it was observed that P. pycnotheca played a key role as primary colonizer of bare soil in areas close to the front of the glacier or around small ponds or temporary streams created after glacier retreat. In these areas, P. pycnotheca sometimes forms dense patches covering hundreds of square meters. In areas where the soil water content has decreased,
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Fig. 1. (A) Zonation commonly observed around small ponds in recently deglaciated areas with a first band (P) of Placopsis pycnotheca close to the waters edge and another band where P. pycnotheca is replaced by bryophytes (B). On the right, the phanerogam Gunnera magellanica forms a dense carpet (G). (B) Soil surface covered almost completely with P. pycnotheca in an area close to the front of the Pía Glacier. (C) Typical rounded thallus of P. pycnotheca showing fusion with smaller thallus in its periphery. Dark orange brown spots are cephalodia. (D) Soil surface covered with scattered P. pycnotheca showing hypothallus contour from an area showing soil erosion. (E) Close-up image of thallus periphery. Arrows point to cephalodia occurring in the thallus periphery before squamules with green photobionts form. (A) Marinelli Glacier, Isla Grande de Tierra del Fuego, Chile; (B)–(E) Pía Glacier, Isla Grande de Tierra del Fuego, Chile. Scale bars: (B) 5 cm; (C) 2.5 cm; (D) 2.5 cm; (E) 5 mm.
P. pycnotheca is replaced by mosses and/or the nitrogen-fixing phanerogam Gunnera magellanica (Fig. 1A). The successful rapid colonization by P. pycnotheca is these areas attracted our interest. We used electron microscopy and gas exchange techniques to investigate the anatomical, morphological and ecophysiological strategies possessed by the lichen.
Materials and methods Sampling area Marinelli Glacier is located in Alberto de Agostini Nacional Park, Isla Grande de Tierra del Fuego, where it descends from the
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Cordillera Darwin and calves into Ainsworth Bay off the Almirantazgo Fjord, 54◦ 32 S 69◦ 34 W. The glacier has been in rapid retreat for several decades. The Pia Glacier also originates on the Darwin Range but flows south to the east arm of Pia Bay, a fjord on the north side of the North-eastern Beagle Channel, approx. 54◦ 46 S 69◦ 40 W. Pia Glacier has also been receding steadily for several decades (Sancho et al., 2011). Samples of P. pycnotheca were collected from beside an ephemeral stream flowing from the Pia Glacier. The soil surface was dry and the lichen thalli could be removed easily as the hyphae bound the sandy substrate together to form coin-like structures about 3–4 cm diameter. The samples were carefully wrapped, placed in plastic bags and held frozen at −20◦ C during their return to the laboratory in Madrid and during subsequent storage. SEM-BSE (scanning electron microscopy–back scattering electron mode) Cross sections of lichen samples were prepared and examined according to the method of Wierzchos and Ascaso (1994). Briefly, the pieces of thalli were first chemically fixed in 2.5% glutaraldehyde in 100 mM phosphate buffer (pH 7.1) and then postfixed in 1% osmium tetroxide in 50 mM sodium phosphate buffer (pH 7.1), dehydrated in a series of ethanol solutions, and gradually infiltrated in LR-White resin. This step was followed by polymerization in an oxygen-free atmosphere (48 h, 60 ◦ C). Blocks of resin-embedded lichen samples were finely polished, first using carborundum of different grades, then sanding papers and silicone carbide and, finally, liquid diamond polishing compound containing diamond particles on a napped cloth. The polished block surfaces were coated with evaporated carbon and observed using a Zeiss DSM 960 SEM microscope equipped with a solid-state, four diodes BSE detector. LTSEM (low temperature SEM) Small fragments of P. pycnotheca were mechanically fixed onto the specimen holder of a cryotransfer system (Oxford CT1500), plunged into subcooled liquid nitrogen, and then transferred to the microscope’s preparation unit via an air-lock transfer device following the protocol described in De los Ríos et al. (1999). The frozen specimens were cryo-fractured in the preparation unit and transferred directly via a second air lock to the microscope cold stage where they were etched for 2 min at −90 ◦ C. After ice sublimation, the etched surfaces were gold sputter coated in the preparation unit and the specimens then placed on the cold stage of the SEM chamber. Fractured surfaces were observed under a DSM 960 Zeiss SEM microscope at −135 ◦ C. CO2 gas exchange CO2 exchange of P. pycnotheca was measured under controlled conditions in the laboratory using an open flow differential IRGA system (CMS 400, Walz, Germany), according to Sancho and Kappen (1989). Before any measurements thalli were pretreated for 2 days at 12 ◦ C, 100 mol photon m−2 s−1 12 h per day and moistened with mineral water (=spring water, composition: bicarbonates 297 mg/ml; sulphates 122 mg/ml; chloride 50.8 mg/ml; calcium 92.3 mg/ml; magnesium 39.6 mg/ml; sodium 33.1 mg/ml) once daily. The response of CO2 exchange to temperature and photon flux density (PFD) was then measured at 0, 5, 10, 15, 20 ◦ C, and 0, 25, 50, 100, 200, 400, 800 and 1200 mol photon m−2 s−1 , respectively. The different PFD were obtained using a KL 2500 LCD lamp (Schott) and a special optic fibre (Walz, Germany) which provides a cold light and avoids heating of the samples inside the cuvette. The response of CO2 exchange to thallus water content was determined by soaking P. pycnotheca thalli in mineral water for 20 min, placing them inside the cuvette at 10 ◦ C and making alternating
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CO2 exchange measurements at 0 and 400 mol photon m−2 s−1 . The supply air humidity range was between 45% and 55% during the drying experiment. The samples were removed and weighed at intervals. An average of three samples was used in all the measurements.
Results Placopsis pycnotheca covered extensive areas of soil in the proximity of the front of the glaciers or around small ponds left after glacier retreatment (Fig. 1A, B). The species grows on sandy to muddy soils, occupying the areas that are closest to water and which can be occasionally flooded. Thalli of P. pycnotheca are made up of a well-defined hypothallus with irregularly rounded surface squamules containing green algae that are more or less separate at first but coalesce in the oldest parts of the thallus (Fig. 1C, D). Squamules on the periphery of the central zone are usually slightly more elongated than in the centre. Beyond the outermost squamules, it is common to observe young tiny squamules and cephalodia (Fig. 1E), the latter often being the first thalline structures formed on the underlying fungal hypothallus. A similar thallus developmental pattern has been described for Rhizocarpon lecanorinum (Clayden, 1998). In contrast to most other lichens, P. pycnotheca colonizes the uppermost millimeters of the soil with mineral particles being included in the thick hypothallus (Fig. 2A), forming a compact crust. In areas where there is some erosion of the soil surface by occasional water flows, rain or possibly even wind, the thalli can appear as small islands because of the protective effect of the hypothallus which is exposed by these processes (Fig. 1D). The net of hyphae (referred in this paper as hypothallus) was easily observable in electron microscopy preparations (Fig. 2A). Squamules had a loose upper cortex and a typical algal layer with numerous algal cells and below, a loose medulla with some algal cells sparsely distributed (Fig. 2A, B). The majority of the thallus was a hypothallus with numerous interspersed soil particles (Fig. 2B) which could reach three times the squamule thickness (700–800 m). Hyphal density was higher in the hypothallus from thalline marginal areas (Fig. 2A) than in central areas (Fig. 2C). LTSEM showed that the hyphae were closely associated to mineral particles, surrounding and embracing them (Fig. 2D and E). Extracellular polymeric substances (EPS) in hydrated thalli were visible with this technique as strands that connected hyphae with other hyphae and mineral particles forming a subsurface network (arrows and asterisks in Fig. 2F). Ice masses were observed by LTSEM within hydrated thalli (asterisks in Fig. 2D) indicating that water can be stored within this crust structure. The ultrastructural appearance of the majority of fungal hyphae indicated that they remain alive deep in the thallus (Fig. 2E). The integrated nature of the thallus is shown by the apparent absence of any other organisms within the medulla and hypothallus. Careful searching in light microscopy preparations found a complete lack of dematiacaeous fungi, and no bacteria, other than cyanobacteria in cephalodia, were detected. Placopsis pycnotheca, like other species of the genus, has abundant cephalodia which can be found throughout the whole thallus (Fig. 3A). They are especially frequent in marginal areas of the thalli (Figs. 1C and 3B), where they seem to appear independently from the squamules. Sometimes, they reached a very large size and were situated under the lobules (Fig. 3C). Incipient cephalodia were often observed up to 600 m below the thallus surface, amongst the mineral particles (arrows in Fig. 3D). It appeared as if they usually occupied areas with scarce mycobiont hyphae but where the liquid water was stored (Fig. 3E). Nostoc-like cyanobionts appeared as rounded cells forming chains. In the early developmental stages, mycobiont hyphae encircled the mucilagenous envelope
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Fig. 2. (A) SEM-BSE (A, B) and LTSEM (C, D, E, F) images of P. pycnotheca thalli. (A) Thallus squamules (S) above a thick hypothallus (H) in which many mineral particles are present. (B) Squamule-hypothallus interface showing the presence of mineral particles of different types. (C) Hydrated P. pycnotheca thallus showing a net of hyphae constituting a thick hypothallus. (D) Detail of hypothallus from hydrated lichen showing mineral particles trapped by fungal hyphae and ice masses (asterisks). (E) Detail of a particle surrounded by fungal hyphae. (F) Presence of EPS associated to fungal hyphae (white arrows) and mineral particles (asterisks).
of the cyanobacteria filaments, forming a clearly separate structure (Fig. 3F). In adult stages, the cephalodia were more compartimentalized with several cyanobiont aggregates within an individual cephalodium, each surrounded and isolated from each other by fungal hyphae, giving the structure a cerebral aspect in longitudinal cuts (Fig. 3A). A similar type of compartmentalized cephalodia has been observed in the genus Lobaria (Jordan, 1970).
Fig. 4 shows the response of net photosynthesis and dark respiration for P. pycnotheca in relation to temperature at optimum WC. As expected, dark respiration increased with temperature from 0.20 mol CO2 m−2 s−1 at 0 ◦ C, to 1.19 mol CO2 m−2 s−1 at 20 ◦ C. At the two highest PFD measured, 800 and 1200 mol photon m−2 s−1 , net photosynthesis also rose with temperature with no maximum being reached. Maximal rates measured at 20 ◦ C were 3.27
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Fig. 3. SEM-BSE (A–D) and LTSEM (E, F) images of P. pycnotheca thalli showing cephalodia (C). (A) Mature cephalodia intermixed with lichen squamules. (B) Marginal part of the thallus containing numerous cephalodia. (C) Large cephalodia under lichen squamules. (D) Incipient cephalodia (arrows) immersed in the hypothallus underneath soil surface. (E) Incipient subsurface cephalodia (c) from hydrated thallus. (F) Detail of subsurface cephalodia observed in (E) showing Nostoc-like cells immersed in a mucilaginous matrix and encircled by mycobiont hyphae.
and 3.94 mol CO2 m−2 s−1 at 800 and 1200 mol photon m−2 s−1 respectively. When measured at lower PFD net photosynthesis declined at higher temperatures as dark respiration increased. Maximal net photosynthesis occurred at around 15 ◦ C at 400 mol photon m−2 s−1 and at 0 ◦ C at 12 mol photon m−2 s−1 . Net photosynthesis rates of water saturated thalli were initially depressed when measured at 10 ◦ C and 400 mol photon m−2 s−1
(Fig. 5). With further drying net photosynthesis increased to a maximal value, close to 2 mol CO2 m−2 s−1 after about 220 min where it remained for about 200 min and then declined due to desiccation. There was still positive net photosynthesis after 700 min (∼ =12 h) drying had elapsed. Extending the period of activity through water storage, as done by P. pycnotheca, means that there is a high probability that the lichen will be active at times of high insolation in its
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Fig. 4. Response of net photosynthesis and dark respiration (mol CO2 m−2 s−1 ) to various combinations of thallus temperature (from 0 to 20 ◦ C) and incident PFD (photosynthetic photon flux density, from 0 to 1200 mol m−2 s−1 ) at optimum water content. Actual PFD used is indicated at the end of each fitted response curve; response curve was fitted using standard polynomial fits.
natural habitat. These will also probably be times of higher thallus temperature. The interaction between net photosynthesis, temperature and PFD is such that at warmer temperatures and high PFD P. pycnotheca achieves its highest net photosynthetic rates. At lower PFD there is an optimum for net photosynthesis at lower temperature so the species achieves good carbon gain at the expected combinations of PFD temperature in the natural habitat. The laboratory studies also suggest that the lichen is well protected against potential damage to the photosystems from high PFD. Discussion Placopsis pycnotheca thalli cover large areas of soil in recently deglaciated areas in Isla Grande of Tierra del Fuego. The thallus structure is unusual in that large amounts of mineral grains are enclosed within a compact and thick hypothallus (rhizohyphae in the sense of Follmann et al., 1991). The hypothallus hyphae play
Fig. 5. Response of net photosynthesis (mol CO2 m−2 s−1 ) of P. pycnotheca after they have been fully hydrated and then allowed to desiccate in the cuvette at 10 ◦ C and 400 mol m−2 s−1 . High thallus water content is therefore to the left and low thallus water content to the right. X-axis is elapsed time in minutes. The curve has been fitted to the results from 3 replicates (R2 = 0.86). Humidity of the air supply during the measurements ranged between 45% and 55%.
an important mechanical role in the trapping of mineral particles but also produce extracellular polysaccharides that cause local binding of mineral particles, and generate a general packing effect (Dorioz et al., 1993). By constructing its thallus in this manner P. pycnotheca can colonize a substrate that would normally be very friable and unstable. The strength of the thallus means that it also resists erosion by rain (Rozzi et al., 2006). P. pycnotheca thalli, therefore, contribute to the early stages of formation of a biological soil crust and protect the fine mineral sands beneath them from erosion by rain, flooding water and wind. More complex early colonizing biological soil crusts including lichens of the Placopsis genus, P. trachyderma, have been described in the braided rivers of southern New Zealand (Ullmann et al., 2007). Our study provides new insights about the formation of cephalodia in Placopsis. Development of cephalodia could start deep within the hypothallus and then extend upwards towards soil surface to occasionally form large cephalodia. Sernander (1907) has described the formation of cephalodia in P. gelida as being independent from the rest of the thallus, and subsequently both parts of the thallus (with green algae and cyanobacteria) merged to form a single thallus. Ott et al. (1997) investigating the ontogeny of cephalodia in five saxicolous species of Placopsis, found species-specific patterns. Whereas in species as P. contortuplicata, P. macrophtalma or P. perrugosa, cephalodia are formed after the thallus is completely differentiated, in species as P. gelida and P. fuscidula cephalodia developed in parallel to the lichen thallus containing green algae. Such differences in timing of cephalodia development had been already reported for other genera as Solorina (Jahns, 1988). Development of cephalodia in P. pycnotheca occurred within the hypothallus but in some parts of the thallus, such as the periphery, cephalodia seem to originate prior to the formation of squamules with green photobionts. Cephalodium primordia can also begin to grow up to 600 m within the hypothallus and do not always reach the thallus surface so that the visible cephalodia are an underestimate of total numbers present. The development of cephalodia in the hypothallus of a saxicolous thallus and their subsequent growth upwards towards thallus surface has already been described in P. contortuplicata and P. gelida (Lamb, 1947; Ott et al., 1997). Schroeter (1994) showed, using chlorophyll a fluorescence techniques, that the cephalodia in Placopsis contortuplicata after wetting with water had substantial electron transport through PSII, CO2 exchange could not be detected when only the cyanobacteria were wetted and it was estimated that the cyanobacteria made no significant contribution to thallus carbon balance. Green et al. (1997) proposed a possible link between nitrogen fixing cephalodia and carbon fixation through more nitrogen being available for constructing photosynthetic machinery, but the link between nitrogen and carbon fixation is still poorly understood. Anatomical features of P. pycnotheca thallus and the associated water holding capacity of the thallus appear to contribute to its pioneer role and growth rates. The thick thallus with its high water storage capacity means that, in the laboratory, the photosynthetic activity is maintained for an unusually long period, around 12 h (Fig. 5). Lange et al. (1994) reported 2.4 h of photosynthetical activity after complete hydration for other soil crust species Lecidella crystallina at 1000 mol photon m−2 s−1 and 20 ◦ C. The saxicolous crustose Lecanora muralis showed around 5.5 h at 1250 mol photon m−2 s−1 and 17 ◦ C (Leisner et al., 1997) and the foliose lichen Peltigera neckeri, 4 h of metabolic activity at 400 mol photon m−2 s−1 and 17 ◦ C (Lange et al., 1996). Under natural conditions the photosynthetic period would probably be even longer as the thallus is connected to water reserves in the moist underlying mineral soil. The lichen showed decreased net photosynthetic rates at higher thallus water content, so-called suprasaturation (Lange et al., 1997, 1999). Maximal depression was about 75% of maximal rates, but
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zero or negative rates were not found. P. pycnotheca was photosynthetically active for about 500 min before decreases due to desiccation occurred but showed decreased net photosynthesis at high water contents for about 200 min (Fig. 5). Although this phenomenon is relatively common in lichens the structural basis for it is still not obvious although it is clearly a result of increased CO2 diffusion resistances in the thallus (Cowan et al., 1992). Lange et al. (1995) showed a very similar performance of net photosynthesis in relation to water content for the soil crust lichen Toninia sedifolia. The good adaptation showed by P. pycnotheca to high irradiances and temperatures in the laboratory indicates that the lichen could perform very well in the field under these conditions. However, it is more likely that photosynthesis in the field is under suboptimal conditions. Periods of concurrent high temperatures, high irradiances and optimum water contents could occur but do not seem to be the prevailing conditions on a humid south-latitude habitat as the study site, and low temperatures and irradiances appear to be much more common during the activity period. The long active period of P. pycnotheca would not only produce high carbon gains, a key point in understanding its fast growing and early colonizer capacity but would also allow extended nitrogen fixation by the cephalodia. Nitrogen fixation would support better growth by the lichen in a region where natural nitrogen sources are very low; atmospheric deposition rates in southern Chile are possibly the lowest in the world (Oyarzun et al., 1998) and the rocks do not offer a supply either. The growth form of this species, constituting an early stage of soil crust where it is embedded in the mineral soil surface, enables it to colonize what would otherwise be a very unstable environment. It also permits it to play a key role in stabilizing the soil and protecting it from both wind and water erosion (Belnap, 2001; Belnap et al., 2001; Follmann et al., 1991) both of which can be very important at the early stages of primary succession in receding glacier areas. The changes induced by this lichen are also probably beneficial for vegetation succession, in particular by improving the nutrient content of the soil and contributing to stabilizing the surface thus leading to the formation of inherently favorable microenvironments important for the establishment of more complex communities in these low-productivity environments (Belnap et al., 2001; Breen and Levesqué, 2006, 2008; De los Ríos et al., 2002; Souza-Egipsy et al., 2004). Acknowledgements The authors thank Fernando Pinto (ICA, CSIC) and Manolo Castillejo and José Manuel Hontoria (MNCN, CSIC) for their technical assistance; Dr. Ricardo Rozzi and Dr. Francisca Massardo and their institutions (Fundación Omora and Universidad de Magallanes) for their scientific supervision in the organization of the field work and their logistic support. Special acknowledgement is due to Captain Mansilla and the crew of the vessel “Don Pelegrín” for their skilful navigation in the highly demanding southern channels and for their kind hospitality on board. This work was supported by grants CTM2009-12838-CO4-01 and CTM2009-12838-CO4-03 from the Spanish Ministry of Science and Innovation. SPO is funded by the program JAE-Doc (Spanish Research Scientific Council). References Belnap, J., 2001. Biological soil crusts and wind erosion. In: Belnap, J., Lange, O.L. (Eds.), Biological Soil Crusts: Structure, Function, and Management. Springer, Berlin-Heidelberg, pp. 339–347. Belnap, J., Büdel, B., Lange, O.L., 2001. Biological soil crusts: characteristics and distribution. In: Belnap, J., Lange, O.L. (Eds.), Biological Soil Crusts: Structure, Function, and Management. Springer, Berlin, Heidelberg, pp. 3–32. Breen, K., Levesqué, E., 2006. Postglacial succession of biological soil crusts and vascular plants: biotic interactions in the High Arctic. Can. J. Bot. 84, 1131–1714.
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Anatomical, morphological and ecophysiological strategies in Placopsis pycnotheca (lichenized fungi, Ascomycota) allowing rapid colonization of recently deglaciated soils Asunción de los Ríos a,∗ , José Raggio b , Sergio Pérez-Ortega a , Mercedes Vivas b , Ana Pintado b , T.G. Allan Green b,c , Carmen Ascaso a , Leopoldo G. Sancho b a
Museo Nacional de Ciencias Naturales, CSIC, C/Serrano 115 dpdo, E-28006 Madrid, Spain Departamento de Biología Vegetal II, Facultad de Farmacia, Universidad Complutense de Madrid, E-28040 Madrid, Spain c Biological Sciences, University of Waikato, Private Bag 3105, Hamilton, New Zealand b
a r t i c l e
i n f o
Article history: Received 11 January 2011 Accepted 20 March 2011 Keywords: Anatomy Cephalodia Gas exchange Receding glaciers Soil crust Terricolous lichen Nitrogen fixation
a b s t r a c t The green algal lichen Placopsis pycnotheca was identified at Pia and Marinelli glaciers (Isla Grande of Tierra de Fuego, Chile) as a primary colonizer of bare soil in areas close to the front of the glacier or around small ponds created after glacier retreatment. Electron microscopy study showed that P. pycnotheca formed a thick hypothallus within which hyphae and their extracellular polymeric substances bind numerous soil particles. This structure augments water holding and soil stabilization capacities and constitutes an early stage in soil crust development. In addition, numerous cephalodia are formed within the hypothallus and subsequently develop upwards towards the thallus surface, sometimes before the formation of squamules with green algae. These anatomical and morphological strategies together with physiological properties such as the long photosynthetic activity period (measured in the laboratory) help explain its pioneering role as a colonizer and its apparently high growth rate. © 2011 Elsevier GmbH. All rights reserved.
Introduction Ecological research analyzing the primary succession on newly exposed soil surfaces after deglaciation is central to advancing our understanding of ecosystem dynamics and for evaluating potential effects of climate warming. Primary succession is generally carried out by microorganisms and cryptogamic organisms that rapidly colonize deglaciated soils well in advance of the vascular plants (Burrows, 1990; Kastovska et al., 2005; Nemergut et al., 2007; Schmidt et al., 2008; Wynn-Williams, 1993). Cyanobacteria, green algae, lichenized and free living fungi, mosses, and heterotrophic bacteria are typically the first organisms to colonize the soil surface and subsurface altering the properties as they form a biological soil crust (Belnap et al., 2001; Breen and Levesqué, 2008; Evans and Johansen, 1999).
∗ Corresponding author. E-mail addresses: [email protected] (A. de los Ríos), [email protected] (J. Raggio), [email protected] (S. Pérez-Ortega), [email protected] (M. Vivas), [email protected] (A. Pintado), [email protected] (T.G.A. Green), [email protected] (C. Ascaso), [email protected] (L.G. Sancho). 0367-2530/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.flora.2011.05.002
The lichen genus Placopsis (Agyriaceae, lichenized Ascomycota) is a widespread early colonizer of rock surfaces and bare soils, such as recently deglaciated areas, especially in subantarctic regions, with 22 species currently known from southern South America (Follmann et al., 1991; Galloway, 2002, 2010). Placopsis species, although having a green algal primary photobiont, also have cyanobacteria in cephalodia and are efficient nitrogen fixers. They, and other cephalodial and cyanobacterial lichens, can contribute to the nitrogen economy in areas where this nutrient is limiting (Breen and Levesqué, 2008). Placopsis pycnotheca I. M. Lamb is known from southern Argentina, the Juan Fernández Archipelago (Chile), from latitude 32◦ S to 54◦ S in the Chilean Andes and from South Georgia (Galloway, 2002, 2010). It is characterized by its crustose terricolous thallus, composed of swollen areoles and squamules, encrusting sand, soil and small pebbles; and by the simple, subglobose to finger-like isidia. During our study of the colonization of recently deglaciated areas in Tierra de Fuego, it was observed that P. pycnotheca played a key role as primary colonizer of bare soil in areas close to the front of the glacier or around small ponds or temporary streams created after glacier retreat. In these areas, P. pycnotheca sometimes forms dense patches covering hundreds of square meters. In areas where the soil water content has decreased,
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Fig. 1. (A) Zonation commonly observed around small ponds in recently deglaciated areas with a first band (P) of Placopsis pycnotheca close to the waters edge and another band where P. pycnotheca is replaced by bryophytes (B). On the right, the phanerogam Gunnera magellanica forms a dense carpet (G). (B) Soil surface covered almost completely with P. pycnotheca in an area close to the front of the Pía Glacier. (C) Typical rounded thallus of P. pycnotheca showing fusion with smaller thallus in its periphery. Dark orange brown spots are cephalodia. (D) Soil surface covered with scattered P. pycnotheca showing hypothallus contour from an area showing soil erosion. (E) Close-up image of thallus periphery. Arrows point to cephalodia occurring in the thallus periphery before squamules with green photobionts form. (A) Marinelli Glacier, Isla Grande de Tierra del Fuego, Chile; (B)–(E) Pía Glacier, Isla Grande de Tierra del Fuego, Chile. Scale bars: (B) 5 cm; (C) 2.5 cm; (D) 2.5 cm; (E) 5 mm.
P. pycnotheca is replaced by mosses and/or the nitrogen-fixing phanerogam Gunnera magellanica (Fig. 1A). The successful rapid colonization by P. pycnotheca is these areas attracted our interest. We used electron microscopy and gas exchange techniques to investigate the anatomical, morphological and ecophysiological strategies possessed by the lichen.
Materials and methods Sampling area Marinelli Glacier is located in Alberto de Agostini Nacional Park, Isla Grande de Tierra del Fuego, where it descends from the
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Cordillera Darwin and calves into Ainsworth Bay off the Almirantazgo Fjord, 54◦ 32 S 69◦ 34 W. The glacier has been in rapid retreat for several decades. The Pia Glacier also originates on the Darwin Range but flows south to the east arm of Pia Bay, a fjord on the north side of the North-eastern Beagle Channel, approx. 54◦ 46 S 69◦ 40 W. Pia Glacier has also been receding steadily for several decades (Sancho et al., 2011). Samples of P. pycnotheca were collected from beside an ephemeral stream flowing from the Pia Glacier. The soil surface was dry and the lichen thalli could be removed easily as the hyphae bound the sandy substrate together to form coin-like structures about 3–4 cm diameter. The samples were carefully wrapped, placed in plastic bags and held frozen at −20◦ C during their return to the laboratory in Madrid and during subsequent storage. SEM-BSE (scanning electron microscopy–back scattering electron mode) Cross sections of lichen samples were prepared and examined according to the method of Wierzchos and Ascaso (1994). Briefly, the pieces of thalli were first chemically fixed in 2.5% glutaraldehyde in 100 mM phosphate buffer (pH 7.1) and then postfixed in 1% osmium tetroxide in 50 mM sodium phosphate buffer (pH 7.1), dehydrated in a series of ethanol solutions, and gradually infiltrated in LR-White resin. This step was followed by polymerization in an oxygen-free atmosphere (48 h, 60 ◦ C). Blocks of resin-embedded lichen samples were finely polished, first using carborundum of different grades, then sanding papers and silicone carbide and, finally, liquid diamond polishing compound containing diamond particles on a napped cloth. The polished block surfaces were coated with evaporated carbon and observed using a Zeiss DSM 960 SEM microscope equipped with a solid-state, four diodes BSE detector. LTSEM (low temperature SEM) Small fragments of P. pycnotheca were mechanically fixed onto the specimen holder of a cryotransfer system (Oxford CT1500), plunged into subcooled liquid nitrogen, and then transferred to the microscope’s preparation unit via an air-lock transfer device following the protocol described in De los Ríos et al. (1999). The frozen specimens were cryo-fractured in the preparation unit and transferred directly via a second air lock to the microscope cold stage where they were etched for 2 min at −90 ◦ C. After ice sublimation, the etched surfaces were gold sputter coated in the preparation unit and the specimens then placed on the cold stage of the SEM chamber. Fractured surfaces were observed under a DSM 960 Zeiss SEM microscope at −135 ◦ C. CO2 gas exchange CO2 exchange of P. pycnotheca was measured under controlled conditions in the laboratory using an open flow differential IRGA system (CMS 400, Walz, Germany), according to Sancho and Kappen (1989). Before any measurements thalli were pretreated for 2 days at 12 ◦ C, 100 mol photon m−2 s−1 12 h per day and moistened with mineral water (=spring water, composition: bicarbonates 297 mg/ml; sulphates 122 mg/ml; chloride 50.8 mg/ml; calcium 92.3 mg/ml; magnesium 39.6 mg/ml; sodium 33.1 mg/ml) once daily. The response of CO2 exchange to temperature and photon flux density (PFD) was then measured at 0, 5, 10, 15, 20 ◦ C, and 0, 25, 50, 100, 200, 400, 800 and 1200 mol photon m−2 s−1 , respectively. The different PFD were obtained using a KL 2500 LCD lamp (Schott) and a special optic fibre (Walz, Germany) which provides a cold light and avoids heating of the samples inside the cuvette. The response of CO2 exchange to thallus water content was determined by soaking P. pycnotheca thalli in mineral water for 20 min, placing them inside the cuvette at 10 ◦ C and making alternating
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CO2 exchange measurements at 0 and 400 mol photon m−2 s−1 . The supply air humidity range was between 45% and 55% during the drying experiment. The samples were removed and weighed at intervals. An average of three samples was used in all the measurements.
Results Placopsis pycnotheca covered extensive areas of soil in the proximity of the front of the glaciers or around small ponds left after glacier retreatment (Fig. 1A, B). The species grows on sandy to muddy soils, occupying the areas that are closest to water and which can be occasionally flooded. Thalli of P. pycnotheca are made up of a well-defined hypothallus with irregularly rounded surface squamules containing green algae that are more or less separate at first but coalesce in the oldest parts of the thallus (Fig. 1C, D). Squamules on the periphery of the central zone are usually slightly more elongated than in the centre. Beyond the outermost squamules, it is common to observe young tiny squamules and cephalodia (Fig. 1E), the latter often being the first thalline structures formed on the underlying fungal hypothallus. A similar thallus developmental pattern has been described for Rhizocarpon lecanorinum (Clayden, 1998). In contrast to most other lichens, P. pycnotheca colonizes the uppermost millimeters of the soil with mineral particles being included in the thick hypothallus (Fig. 2A), forming a compact crust. In areas where there is some erosion of the soil surface by occasional water flows, rain or possibly even wind, the thalli can appear as small islands because of the protective effect of the hypothallus which is exposed by these processes (Fig. 1D). The net of hyphae (referred in this paper as hypothallus) was easily observable in electron microscopy preparations (Fig. 2A). Squamules had a loose upper cortex and a typical algal layer with numerous algal cells and below, a loose medulla with some algal cells sparsely distributed (Fig. 2A, B). The majority of the thallus was a hypothallus with numerous interspersed soil particles (Fig. 2B) which could reach three times the squamule thickness (700–800 m). Hyphal density was higher in the hypothallus from thalline marginal areas (Fig. 2A) than in central areas (Fig. 2C). LTSEM showed that the hyphae were closely associated to mineral particles, surrounding and embracing them (Fig. 2D and E). Extracellular polymeric substances (EPS) in hydrated thalli were visible with this technique as strands that connected hyphae with other hyphae and mineral particles forming a subsurface network (arrows and asterisks in Fig. 2F). Ice masses were observed by LTSEM within hydrated thalli (asterisks in Fig. 2D) indicating that water can be stored within this crust structure. The ultrastructural appearance of the majority of fungal hyphae indicated that they remain alive deep in the thallus (Fig. 2E). The integrated nature of the thallus is shown by the apparent absence of any other organisms within the medulla and hypothallus. Careful searching in light microscopy preparations found a complete lack of dematiacaeous fungi, and no bacteria, other than cyanobacteria in cephalodia, were detected. Placopsis pycnotheca, like other species of the genus, has abundant cephalodia which can be found throughout the whole thallus (Fig. 3A). They are especially frequent in marginal areas of the thalli (Figs. 1C and 3B), where they seem to appear independently from the squamules. Sometimes, they reached a very large size and were situated under the lobules (Fig. 3C). Incipient cephalodia were often observed up to 600 m below the thallus surface, amongst the mineral particles (arrows in Fig. 3D). It appeared as if they usually occupied areas with scarce mycobiont hyphae but where the liquid water was stored (Fig. 3E). Nostoc-like cyanobionts appeared as rounded cells forming chains. In the early developmental stages, mycobiont hyphae encircled the mucilagenous envelope
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Fig. 2. (A) SEM-BSE (A, B) and LTSEM (C, D, E, F) images of P. pycnotheca thalli. (A) Thallus squamules (S) above a thick hypothallus (H) in which many mineral particles are present. (B) Squamule-hypothallus interface showing the presence of mineral particles of different types. (C) Hydrated P. pycnotheca thallus showing a net of hyphae constituting a thick hypothallus. (D) Detail of hypothallus from hydrated lichen showing mineral particles trapped by fungal hyphae and ice masses (asterisks). (E) Detail of a particle surrounded by fungal hyphae. (F) Presence of EPS associated to fungal hyphae (white arrows) and mineral particles (asterisks).
of the cyanobacteria filaments, forming a clearly separate structure (Fig. 3F). In adult stages, the cephalodia were more compartimentalized with several cyanobiont aggregates within an individual cephalodium, each surrounded and isolated from each other by fungal hyphae, giving the structure a cerebral aspect in longitudinal cuts (Fig. 3A). A similar type of compartmentalized cephalodia has been observed in the genus Lobaria (Jordan, 1970).
Fig. 4 shows the response of net photosynthesis and dark respiration for P. pycnotheca in relation to temperature at optimum WC. As expected, dark respiration increased with temperature from 0.20 mol CO2 m−2 s−1 at 0 ◦ C, to 1.19 mol CO2 m−2 s−1 at 20 ◦ C. At the two highest PFD measured, 800 and 1200 mol photon m−2 s−1 , net photosynthesis also rose with temperature with no maximum being reached. Maximal rates measured at 20 ◦ C were 3.27
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Fig. 3. SEM-BSE (A–D) and LTSEM (E, F) images of P. pycnotheca thalli showing cephalodia (C). (A) Mature cephalodia intermixed with lichen squamules. (B) Marginal part of the thallus containing numerous cephalodia. (C) Large cephalodia under lichen squamules. (D) Incipient cephalodia (arrows) immersed in the hypothallus underneath soil surface. (E) Incipient subsurface cephalodia (c) from hydrated thallus. (F) Detail of subsurface cephalodia observed in (E) showing Nostoc-like cells immersed in a mucilaginous matrix and encircled by mycobiont hyphae.
and 3.94 mol CO2 m−2 s−1 at 800 and 1200 mol photon m−2 s−1 respectively. When measured at lower PFD net photosynthesis declined at higher temperatures as dark respiration increased. Maximal net photosynthesis occurred at around 15 ◦ C at 400 mol photon m−2 s−1 and at 0 ◦ C at 12 mol photon m−2 s−1 . Net photosynthesis rates of water saturated thalli were initially depressed when measured at 10 ◦ C and 400 mol photon m−2 s−1
(Fig. 5). With further drying net photosynthesis increased to a maximal value, close to 2 mol CO2 m−2 s−1 after about 220 min where it remained for about 200 min and then declined due to desiccation. There was still positive net photosynthesis after 700 min (∼ =12 h) drying had elapsed. Extending the period of activity through water storage, as done by P. pycnotheca, means that there is a high probability that the lichen will be active at times of high insolation in its
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Fig. 4. Response of net photosynthesis and dark respiration (mol CO2 m−2 s−1 ) to various combinations of thallus temperature (from 0 to 20 ◦ C) and incident PFD (photosynthetic photon flux density, from 0 to 1200 mol m−2 s−1 ) at optimum water content. Actual PFD used is indicated at the end of each fitted response curve; response curve was fitted using standard polynomial fits.
natural habitat. These will also probably be times of higher thallus temperature. The interaction between net photosynthesis, temperature and PFD is such that at warmer temperatures and high PFD P. pycnotheca achieves its highest net photosynthetic rates. At lower PFD there is an optimum for net photosynthesis at lower temperature so the species achieves good carbon gain at the expected combinations of PFD temperature in the natural habitat. The laboratory studies also suggest that the lichen is well protected against potential damage to the photosystems from high PFD. Discussion Placopsis pycnotheca thalli cover large areas of soil in recently deglaciated areas in Isla Grande of Tierra del Fuego. The thallus structure is unusual in that large amounts of mineral grains are enclosed within a compact and thick hypothallus (rhizohyphae in the sense of Follmann et al., 1991). The hypothallus hyphae play
Fig. 5. Response of net photosynthesis (mol CO2 m−2 s−1 ) of P. pycnotheca after they have been fully hydrated and then allowed to desiccate in the cuvette at 10 ◦ C and 400 mol m−2 s−1 . High thallus water content is therefore to the left and low thallus water content to the right. X-axis is elapsed time in minutes. The curve has been fitted to the results from 3 replicates (R2 = 0.86). Humidity of the air supply during the measurements ranged between 45% and 55%.
an important mechanical role in the trapping of mineral particles but also produce extracellular polysaccharides that cause local binding of mineral particles, and generate a general packing effect (Dorioz et al., 1993). By constructing its thallus in this manner P. pycnotheca can colonize a substrate that would normally be very friable and unstable. The strength of the thallus means that it also resists erosion by rain (Rozzi et al., 2006). P. pycnotheca thalli, therefore, contribute to the early stages of formation of a biological soil crust and protect the fine mineral sands beneath them from erosion by rain, flooding water and wind. More complex early colonizing biological soil crusts including lichens of the Placopsis genus, P. trachyderma, have been described in the braided rivers of southern New Zealand (Ullmann et al., 2007). Our study provides new insights about the formation of cephalodia in Placopsis. Development of cephalodia could start deep within the hypothallus and then extend upwards towards soil surface to occasionally form large cephalodia. Sernander (1907) has described the formation of cephalodia in P. gelida as being independent from the rest of the thallus, and subsequently both parts of the thallus (with green algae and cyanobacteria) merged to form a single thallus. Ott et al. (1997) investigating the ontogeny of cephalodia in five saxicolous species of Placopsis, found species-specific patterns. Whereas in species as P. contortuplicata, P. macrophtalma or P. perrugosa, cephalodia are formed after the thallus is completely differentiated, in species as P. gelida and P. fuscidula cephalodia developed in parallel to the lichen thallus containing green algae. Such differences in timing of cephalodia development had been already reported for other genera as Solorina (Jahns, 1988). Development of cephalodia in P. pycnotheca occurred within the hypothallus but in some parts of the thallus, such as the periphery, cephalodia seem to originate prior to the formation of squamules with green photobionts. Cephalodium primordia can also begin to grow up to 600 m within the hypothallus and do not always reach the thallus surface so that the visible cephalodia are an underestimate of total numbers present. The development of cephalodia in the hypothallus of a saxicolous thallus and their subsequent growth upwards towards thallus surface has already been described in P. contortuplicata and P. gelida (Lamb, 1947; Ott et al., 1997). Schroeter (1994) showed, using chlorophyll a fluorescence techniques, that the cephalodia in Placopsis contortuplicata after wetting with water had substantial electron transport through PSII, CO2 exchange could not be detected when only the cyanobacteria were wetted and it was estimated that the cyanobacteria made no significant contribution to thallus carbon balance. Green et al. (1997) proposed a possible link between nitrogen fixing cephalodia and carbon fixation through more nitrogen being available for constructing photosynthetic machinery, but the link between nitrogen and carbon fixation is still poorly understood. Anatomical features of P. pycnotheca thallus and the associated water holding capacity of the thallus appear to contribute to its pioneer role and growth rates. The thick thallus with its high water storage capacity means that, in the laboratory, the photosynthetic activity is maintained for an unusually long period, around 12 h (Fig. 5). Lange et al. (1994) reported 2.4 h of photosynthetical activity after complete hydration for other soil crust species Lecidella crystallina at 1000 mol photon m−2 s−1 and 20 ◦ C. The saxicolous crustose Lecanora muralis showed around 5.5 h at 1250 mol photon m−2 s−1 and 17 ◦ C (Leisner et al., 1997) and the foliose lichen Peltigera neckeri, 4 h of metabolic activity at 400 mol photon m−2 s−1 and 17 ◦ C (Lange et al., 1996). Under natural conditions the photosynthetic period would probably be even longer as the thallus is connected to water reserves in the moist underlying mineral soil. The lichen showed decreased net photosynthetic rates at higher thallus water content, so-called suprasaturation (Lange et al., 1997, 1999). Maximal depression was about 75% of maximal rates, but
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zero or negative rates were not found. P. pycnotheca was photosynthetically active for about 500 min before decreases due to desiccation occurred but showed decreased net photosynthesis at high water contents for about 200 min (Fig. 5). Although this phenomenon is relatively common in lichens the structural basis for it is still not obvious although it is clearly a result of increased CO2 diffusion resistances in the thallus (Cowan et al., 1992). Lange et al. (1995) showed a very similar performance of net photosynthesis in relation to water content for the soil crust lichen Toninia sedifolia. The good adaptation showed by P. pycnotheca to high irradiances and temperatures in the laboratory indicates that the lichen could perform very well in the field under these conditions. However, it is more likely that photosynthesis in the field is under suboptimal conditions. Periods of concurrent high temperatures, high irradiances and optimum water contents could occur but do not seem to be the prevailing conditions on a humid south-latitude habitat as the study site, and low temperatures and irradiances appear to be much more common during the activity period. The long active period of P. pycnotheca would not only produce high carbon gains, a key point in understanding its fast growing and early colonizer capacity but would also allow extended nitrogen fixation by the cephalodia. Nitrogen fixation would support better growth by the lichen in a region where natural nitrogen sources are very low; atmospheric deposition rates in southern Chile are possibly the lowest in the world (Oyarzun et al., 1998) and the rocks do not offer a supply either. The growth form of this species, constituting an early stage of soil crust where it is embedded in the mineral soil surface, enables it to colonize what would otherwise be a very unstable environment. It also permits it to play a key role in stabilizing the soil and protecting it from both wind and water erosion (Belnap, 2001; Belnap et al., 2001; Follmann et al., 1991) both of which can be very important at the early stages of primary succession in receding glacier areas. The changes induced by this lichen are also probably beneficial for vegetation succession, in particular by improving the nutrient content of the soil and contributing to stabilizing the surface thus leading to the formation of inherently favorable microenvironments important for the establishment of more complex communities in these low-productivity environments (Belnap et al., 2001; Breen and Levesqué, 2006, 2008; De los Ríos et al., 2002; Souza-Egipsy et al., 2004). Acknowledgements The authors thank Fernando Pinto (ICA, CSIC) and Manolo Castillejo and José Manuel Hontoria (MNCN, CSIC) for their technical assistance; Dr. Ricardo Rozzi and Dr. Francisca Massardo and their institutions (Fundación Omora and Universidad de Magallanes) for their scientific supervision in the organization of the field work and their logistic support. Special acknowledgement is due to Captain Mansilla and the crew of the vessel “Don Pelegrín” for their skilful navigation in the highly demanding southern channels and for their kind hospitality on board. This work was supported by grants CTM2009-12838-CO4-01 and CTM2009-12838-CO4-03 from the Spanish Ministry of Science and Innovation. SPO is funded by the program JAE-Doc (Spanish Research Scientific Council). References Belnap, J., 2001. Biological soil crusts and wind erosion. In: Belnap, J., Lange, O.L. (Eds.), Biological Soil Crusts: Structure, Function, and Management. Springer, Berlin-Heidelberg, pp. 339–347. Belnap, J., Büdel, B., Lange, O.L., 2001. Biological soil crusts: characteristics and distribution. In: Belnap, J., Lange, O.L. (Eds.), Biological Soil Crusts: Structure, Function, and Management. Springer, Berlin, Heidelberg, pp. 3–32. Breen, K., Levesqué, E., 2006. Postglacial succession of biological soil crusts and vascular plants: biotic interactions in the High Arctic. Can. J. Bot. 84, 1131–1714.
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