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Unicellular green algae of the genus Interfilum (Klebsormidiales, Streptophyta) are typical components of biological soil crusts. Four different aeroterrestrial Interfilum strains that have previously been molecular-taxonomically characterized and isolated from temperate soils in Belgium, Czech Republic, New Zealand, and Ukraine were investigated. Photosynthetic performance was evaluated under different controlled abiotic conditions, including dehydration, as well as under a light and temperature gradient. For standardized desiccation experiments, a new methodological approach with silica gel filled polystyrol boxes and effective quantum yield measurements from the outside were successfully applied. All Interfilum isolates showed a decrease and inhibition of the effective quantum yield under this treatment, however with different kinetics. While the single cell strains exhibited relatively fast inhibition, the cell packet forming isolates dried slower. Most strains fully recovered effective quantum yield after rehydration. All Interfilum isolates exhibited optimum photosynthesis at low photon fluence rates, but with no indication of photoinhibition under high light conditions suggesting flexible acclimation mechanisms of the photosynthetic machinery. Photosynthesis under lower temperatures was generally more active than respiration, while the opposite was true for higher temperatures. The presented data provide an explanation for the regular occurrence of Interfilum species in soil habitats where environmental factors can be particularly harsh.
While many green algae occur under freshwater and marine conditions, they are not restricted to these aquatic systems. Green algae may also be found in diverse types of terrestrial environments, such as on and under rocks, on plant surfaces or in association with soils (Ettl and Gärtner 1995). Only hydrated cells will be physiologically active (Häubner et al. 2006, Gray et al. 2007, Karsten et al. 2007). In aquatic ecosystems, most algae are only rarely confronted with dehydration and are potentially capable of being continuously metabolically active. In contrast, aeroterrestrial algae often experience water loss, and hence they can be metabolically active only when there is sufficient water supply. Dehydration strongly affects photosynthesis and growth and has destructive effects on the ultrastructure of the cells (Holzinger and Karsten 2013). In temperate regions, water supply changes with the course of the seasons from humid periods with rain, dew, or snow to extended periods of drought or freezing. Water supply is therefore the key ecological factor for long-term survival of aeroterrestrial green algae (Holzinger and Karsten 2013). Besides the effects by fluctuating meteorological conditions on the water status of the cell, intrinsic factors like cell wall properties, extracellular polymeric substances (EPS), formation of cell aggregates, or the biochemical capability to synthesize osmotically active substances represent an important mechanism for algal cells to hold water (Yancey 2005, Flechtner 2007, Rindi 2007). In addition, aeroterrestrial algae are often exposed to other harsh environmental conditions in comparison to their aquatic relatives, such as fluctuating temperatures and high insolation (photosynthetic active radiation [PAR] and UV; Gray et al. 2007). Therefore, aeroterrestrial algae have to be well adapted to these often extreme physical factors to guarantee long-term survival in terrestrial environments.
Recently, the evolution and phylogeny of green algae was critically reviewed by Leliaert et al. (2012 and references therein). These authors showed that the Viridiplantae is comprised of two discrete lineages, the Chlorophyta and Streptophyta. The Chlorophyta consists of several classes including the Trebouxiophyceae, which contains many aeroterrestrial taxa (Friedl and Rybalka 2012). The other lineage, the Streptophyta, includes the charophytes from which the land plants evolved, and which is considered as an older phylum compared to land plants (Leliaert et al. 2012). Typical aeroterrestrial representatives of the streptophytan lineage are members of the Klebsormidiophyceae, that include the genera Klebsormidium and Interfilum, which form unbranched filaments, unicellular aggregations, cell packets, or sarcinoid colonies (Mikhailyuk et al. 2008, Sluiman et al. 2008, Rindi et al. 2011).
Interfilum R.Chodat species are considered important and widely distributed components in various terrestrial habitats, particularly on soils or in association with biological soil crusts (Mikhailyuk et al. 2008, Rindi et al. 2011, and references therein). Many reports of their distribution suggest that they originated from Europe, but Interfilum has also been described in recent years from Argentina (Ehrenhaus and Vigna 2008), New Zealand (Novis and Visnovsky 2011), and Antarctica (Worland and Lukešová 2000), indicating that members of this genus have probably a worldwide distribution similar to the closely related Klebsormidium (Rindi et al. 2011). Molecular data of the New Zealand isolate of Interfilum terricola (Novis and Visnovsky 2011) are in close agreement with those of this species from Belgium (SAG 2100) (Mikhailyuk et al. 2008). Consequently, Novis and Visnovsky (2011) concluded that based on these genetic data, diversification has not occurred in isolation in New Zealand, and that global dispersal of Interfilum must occur quite readily, with no detectable difference from European strains. Closely related Klebsormidium strains can be successfully established as cultures from airborne-samples collected from aircraft in flight (Brown et al. 1964), supporting the concept of ease of dispersal. Sharma et al. (2007) indicate that typical aeroterrestrial green algal taxa such as Klebsormidium (and probably Interfilum) can be dispersed as single cells, filaments (or fragments of filaments), and/or spores via atmospheric transport, and hence it is reasonable to assume that colonization of new terrestrial surfaces such as soil is mediated by such airborne algal cells or spores. However, Interfilum has not yet been reported for many geographic regions. The reason could be simply related to the fact that the identification of unicellular and sarcinoid green algae generally presents a challenge due to the scarcity of visual characters that are available for diagnostic purposes (Mikhailyuk et al. 2008).
Along with other microorganisms such as bacteria, cyanobacteria, and fungi, as well as with macroscopic lichens and bryophytes, Interfilum represents an important phototrophic component of biological soil crusts (Mikhailyuk et al. 2008). These communities produce a joint matrix by gluing soil particles to themselves, thereby forming productive microbial biomasses in the “earth critical zone” which represents the uppermost ~10 mm of soils in many drylands (Belnap and Lange 2001, Pointing and Belnap 2012). Such microbiotic crusts exert a dominating influence on global carbon fixation (~7% of terrestrial vegetation) and nitrogen fixation (~50% of terrestrial biological N fixation; Elbert et al. 2012), along with other ecological functions such as mineralization, water retention, stabilization of soils, and dust trapping (Evans and Johansen 1999, Reynolds et al. 2001, Lewis 2007, Castillo-Monroy et al. 2010).
Although the ecological roles of Klebsormidiales are considered significant, only a few studies exist on their ecophysiological response patterns as a function of environmental stress scenarios. While this lack of knowledge has been at least partly addressed in recent years for Klebsormidium from urban and alpine habitats (Karsten and Rindi 2010, Karsten et al. 2010, Holzinger et al. 2011, Kaplan et al. 2012, Karsten and Holzinger 2012), Interfilum is still completely unstudied.
In the present study, we examined for the first time the ecophysiological performance of four different strains of Interfilum from temperate soils in Belgium, Czech Republic, Ukraine, and New Zealand. These sites exhibit a gradient of precipitation ranging from 388 mm (Karadag, Crimea, Ukraine) to 1,162 mm annual rainfall (Waroneu, Belgium; www.worldclimate.com). According to Rindi et al. (2011) this genus forms the superclade A in his molecular-phylogenetic treatment of Klebsormidium/Interfilum, and the four investigated Interfilum isolates were assigned to subclade A1 (Interfilum massjukiae), A2 (I. terricola), A3 (Interfilum sp.), and A4 (Interfilum sp.). While I. massjukiae and I. terricola represent morphologically well described species, A3 and A4, the two earliest-diverging subclades, could not be identified unambiguously, and hence may represent undescribed taxa (Mikhailyuk et al. 2008, Rindi et al. 2011). The four Interfilum strains exhibited different morphologies ranging from single cells to cell packets in combination with the occurrence or lack of mucilage.
The physiological parameters (i.e., dehydration and temperature tolerance) and the light requirements for photosynthesis were investigated under the same conditions in the four Interfilum strains. The main goal of this study was to evaluate whether morphology and mucilage influence dehydration dynamics and the physiology of the organisms. Of particular interest was to consider whether the precipitation features of the original habitat are reflected in the respective response patterns.
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Packet-like aggregation of cells is typical for many terrestrial green algae from different phylogenetic lineages (Lopez-Bautista et al. 2008, Rindi et al. 2009). The most widespread and successful terrestrial unicellular algal taxa include the genera Apatococcus, Chlorosarcinopsis, Chlorosarcina, Chlorokybus, Desmococcus, Trebouxia, Tetracystis, and Interfilum (Hoffmann 1989, Lopez-Bautista et al. 2008, Rindi et al. 2011). The cell “self-protection” in these aggregates is considered as adaptation of algal cells to retain cellular water (Nienow 1996). Recent investigation of freshwater Coleochete species under simulated terrestrial culture conditions showed a strong change in morphology from typical radial thallus to the formation of packet-like structures (Graham et al. 2012). Therefore, the formation of a packet-like morphotype might be a general strategy of algae to thrive under terrestrial conditions.
From an ecological view, the differences in the dehydration response kinetics of the four Interfilum strains also reflected meteorological data in the respective natural habitat. I. terricola SAG 2100 and Interfilum sp. SAG 36.88 (Table 1) were isolated from soils under oceanic, humid influence, which may explain their higher susceptibility against water loss. Both collecting locations in Belgium and New Zealand are characterized by a marine coastal climate that typically is mild with no dry season, warm summers, and moderate seasonality (Köppen-Geiger classification: Cfb; Peel et al. 2007). In contrast, I. massjukiae SAG 2102 and Interfilum sp. SAG 2147 (Table 1) were collected from drier habitats with continental warm summers (Köppen-Geiger classification: Dfb; Peel et al. 2007). The Interfilum strains maintained in culture under identical abiotic conditions for long periods still exhibited conspicuous differences in morphology, aggregation, mucilage formation, and ecophysiological response patterns, which indicates that these aeroterrestrial algae did not lose their genetic traits.
Recovery kinetics of after rehydration of the dried Interfilum samples also showed conspicuous interspecific differences. While I. terricola SAG 2100, I. massjukiae SAG 2102, and Interfilum sp. SAG 2147 fully recovered upon rehydration, although after different time intervals, Interfilum sp. SAG 36.88 only partly recovered (60% of control) which points to some damage in the photosynthetic apparatus. In the microscopic observations, this strain appeared to be the most fragile with a higher surface to volume ratio when compared to the elliptic cells of the other strains. In another study, the optimum quantum yield as a function of changing water availability was followed over several months on an aeroterrestrial green algal biofilm growing on anthropogenic hard substrata (Häubner et al. 2006). This biofilm, which was dominated by unicellular taxa, was controlled by dew; with increasing insolation after sunrise, algal cells continuously lost water resulting in inhibition of photosynthesis. After artificial moistening, however, quick recovery of photosynthesis was observed (Häubner et al. 2006), which was similar to the recovery kinetics in Interfilum strains analyzed in the present study. Such high dehydration tolerance in conjunction with high recovery rates seems to be a typical feature of many aeroterrestrial green algae from dune, alpine, and dryland soil crust communities (De Winder et al. 1990, Gray et al. 2007, Karsten et al. 2010, Karsten and Holzinger 2012).
The mechanisms involved in desiccation tolerance of green algae such as Interfilum or Klebsormidium are still poorly understood. In general, desiccation-tolerant plants can be divided into two groups according to Oliver and Bewley (1997): those classified as “modified desiccation-tolerant plants” (some “higher plants”, i.e., resurrection plants) and the “fully desiccation-tolerant plants” (some bryophytes, small pteridophytes, and aeroterrestrial algae). Tolerant “higher plants” only survive if dehydration is moderate and very slow, so that full recovery is guaranteed. The main strategy of these plants is to induce protective mechanisms during desiccation, rather than repair upon rehydration (Alpert and Oliver 2002). In contrast, aeroterrestrial algae and cyanobacteria can survive very rapid drying events for a long time and quickly recover after rehydration (Potts 2001). However, molecular and biochemical mechanisms involved in desiccation tolerance of “higher” and “lower plants” have not been investigated so far in Interfilum and Klebsormidium.
Aeroterrestrial green algae are confronted with problems in carbon dioxide supply for photosynthesis as a function of their cellular water status, since the diffusion of this gas is almost 9,000-fold higher in air as in water (Green et al. 2008). Consequently, the precise water status of Interfilum should ideally always be known during experimentation, but this is methodologically challenging, and hence could not be considered in the present study. In Klebsormidium flaccidum (De Winder et al. 1990) and various lichens (Green et al. 2008), it could be shown that strong loss of water resulted in depression of photosynthesis if species-specific cell water concentrations go below this value, which is in principle confirmed by the Interfilum data. In addition, too much water, e.g., after strong precipitation, had a negative effect on photosynthesis in lichens. The inhibited photosynthesis under these conditions was related to the establishment of an external water film, which acted as a barrier by reducing carbon dioxide diffusion from the air into the algal cells (Green et al. 2008).
Measurements of PI curves enable the light requirements of algae for photosynthesis to be characterized, particularly in the context of fluctuating radiation conditions (Henley 1993). High α together with low Ic and Ik values, as measured in the four Interfilum strains, are typical for algae living under shaded conditions. Although microclimatic information on the natural habitats of the Interfilum species are missing, we know from our field observations on Klebsormidium and from Dr. T. Mikhailyuk (pers comm), that it appears to prefer low radiation conditions such as under the canopy of oak trees (I. terricola SAG 2100; Table 1). However, in the present study PAM measurements under enhanced photon fluence rates clearly documented that all Interfilum isolates were not photoinhibited. This is a rather unusual photophysiological response pattern, as in most algae studied so far low radiation adaptation of photosynthesis is typically reflected in photoinhibition under high light conditions (e.g., Bischof et al. 1998). From the few data available on Klebsormidium, it seems that such a photophysiological plasticity is a common trait for soil crust green algae to better cope with strong fluctuating changes in the natural light climate (Kaplan et al. 2012, Karsten and Holzinger 2012). In “higher plants” and various algal groups, a set of mechanisms have been described which limit the extent of photodamage and/or which efficiently repair photodamage (Raven 2011). These mechanisms include partial avoidance of photodamage by restricting the number of photons incident on the photosynthetic apparatus. For example, phototactic movement of plastids within cells to minimize the absorption of incident PAR. Other avoiding mechanisms typically aim to dissipate excitation of photosynthetic pigments, e.g., by nonphotochemical quenching (e.g., xanthophyll cycle) or photochemical quenching (e.g., alternative electron transport pathways) (Raven 2011). The reason for the lack of photoinhibition in all Interfilum isolates might be explained by one of these mechanisms, but needs to be further investigated.
The effect of increasing temperatures on respiratory oxygen consumption and photosynthetic oxygen production in the four Interfilum isolates showed intraspecific, as well as strong differences in the temperature requirements of both physiological processes. While three of four Interfilum strains exhibited optimum photosynthesis between 30°C and 40°C, one isolate was already strongly inhibited under these temperatures, and at 45°C none of the isolates showed oxygen evolution. In contrast, respiration of all strains was not detectable at low temperatures, and was highest between 35°C and 45°C. The emerging picture is that photosynthesis under lower temperatures is generally more efficiently functioning than respiration, while the opposite is true for higher temperatures, where respiration typically exhibits enhanced activity rates compared to photosynthesis. This is also in agreement with the P:R ratios (Fig. 7), which confirm a high net carbon gain and hence biomass formation mainly under temperate conditions, thereby reflecting the natural habitats of the four Interfilum strains. Consequently, the physiological requirements point to optimum in situ photosynthesis activity periods of Interfilum during cool and moist spring and autumn in temperate regions compared to the often rather dry and warm summer months (http://www.worldclimate.com). This disproportionate effect of temperature on two of the key physiological processes in aeroterrestrial green algae has also been documented in the closely related Klebsormidium crenulatum and Klebsormidium dissectum (Karsten et al. 2010, Karsten and Holzinger 2012), as well as in Prasiola crispa from Antarctica (Davey 1989). The latter species is a macroalgal member of the Trebouxiophyceae (Chlorophyta), and hence from an evolutionary standpoint far distant from Interfilum and Klebsormidium. Nevertheless, similar physiological response patterns with rising temperatures strongly support the assumption that aeroterrestrial green algae in general, and independent of their phylogenetic position, exhibit this trait. The conspicuously different temperature requirements for photosynthesis and respiration can be explained by the fact that the first process is more dependent on light-related processes (light absorption, energy transfer etc.) than on temperature, while the second one is completely controlled by temperature (Atkin and Tjoelker 2003). Algal respiration consists of a set of catabolic reactions, localized in different cellular compartments and controlled by a whole array of specific enzymes, of which many exhibit different temperature optima. One partly inhibited respiratory enzyme under cool conditions would be sufficient to act as a bottleneck affecting the whole process (Atkin and Tjoelker 2003).
In conclusion, the aeroterrestrial Interfilum isolates investigated for the first time in the present study, exhibited a high dehydration tolerance, low light requirements for photosynthesis in combination with a lack of photoinhibition, and a relatively high temperature tolerance compared to the monthly maximum air temperature of the respective habitats (Table 1), which concur with the regular occurrence in temperate soils or temperate biological soil crust communities. Although water availability seems to be the ecological key factor for aeroterrestrial algae, there exists of course under natural conditions an interrelationship of all environmental factors, which affect and control the optimum water content in terrestrial habitats. However, further information on inter- and infraspecific differentiation in Interfilum is urgently needed, to better understand physiological plasticity and adaptive strategies of this genus along with state-of-the-art approaches in genomics and transcriptomics.