Terrestrial Green Algae Show Higher Tolerance to Dehydration than Do Their Aquatic Sister-Species

Abstract

Diverse algae possess the ability to recover from extreme desiccation without forming specialized resting structures. Green algal genera such as Tetradesmus (Sphaeropleales, Chlorophyceae) contain temperate terrestrial, desert, and aquatic species, providing an opportunity to compare physiological traits associated with the transition to land in closely related taxa. We subjected six species from distinct habitats to three dehydration treatments varying in relative humidity (RH 5%, 65%, 80%) followed by short- and long-term rehydration. We tested the capacity of the algae to recover from dehydration using the effective quantum yield of photosystem II as a proxy for physiological activity. The degree of recovery was dependent both on the habitat of origin and the dehydration scenario, with terrestrial, but not aquatic, species recovering from dehydration. Distinct strains of each species responded similarly to dehydration and rehydration, with the exception of one aquatic strain that recovered from the mildest dehydration treatment. Cell ultrastructure was uniformly maintained in both aquatic and desert species during dehydration and rehydration, but staining with an amphiphilic styryl dye indicated damage to the plasma membrane from osmotically induced water loss in the aquatic species. These analyses demonstrate that terrestrial Tetradesmus possess a vegetative desiccation tolerance phenotype, making these species ideal for comparative omics studies.

Introduction

Vegetative desiccation tolerance is the ability of organisms to recover from extreme cellular water loss (i.e., final water content below 10% or 0.1 g H₂O per gram dry weight) without forming specialized dormant structures [1]. Such a water content is usually achieved by equilibrating to the water potential of the surrounding dry air with a relative humidity (RH) below 50% [2, 3]. Some desiccation-tolerant plants can survive only short periods of desiccation or require desiccation to onset slowly. Others, including some species of algae, bryophytes, and lycophytes, restore their physiological activity even after rapid or lengthy desiccation (e.g., [4]). Vegetative desiccation tolerance is thought to be one of the key traits in the evolution of early land plants as they evolved from aquatic ancestors [5], and considerable research has focused on advancing our understanding of this capacity in mosses, lycophytes, and their immediate algal relatives. This research showed that multiple cellular processes are affected by desiccation including carbon, protein, and lipid metabolism, cell cycle, stress response pathways, and signaling activity (e.g., [4–10]).

Our current understanding of the mechanisms and evolution of vegetative desiccation tolerance in land plants is mostly based on the comparison of a single terrestrial lineage of land plants (embryophytes) with streptophyte green algae [6, 11], a combined divergence that spans 900+ MY [12]. Unlike the land plants, which evolved on land once, green algae show multiple origins of terrestriality, including lineages inhabiting extreme environments, such as deserts [13–15], providing multiple opportunities to understand and characterize the array of adaptations that may facilitate desiccation tolerance. In deserts, the surface of the soil can be covered by soil crusts, a complex community of microorganisms, fungi, and bryophytes that together act as ecosystem engineers by powering nutrient cycles, preventing erosion, and enhancing water holding capacity, as well as influencing the composition of plant communities [16–18]. Of the bryophyte members of desert soil crusts, many possess cellular mechanisms allowing them to withstand desiccation events and persist in their environment (e.g., [8, 19]). The specific mechanisms of desiccation tolerance across desert algal species are not as well studied, but it is clear that these taxa can recover even after long and extreme desiccation [20].

Desert green algae arose multiple times during the diversification of Chlorophyta, often in clades containing both aquatic and terrestrial species [10, 13, 21, 22]. For example, Tetradesmus G.M. Smith (Sphaeropleales, Chlorophyceae, Chlorophyta), a common planktonic freshwater genus of unicellular or colonial green algae, contains temperate and desert soil species that are closely related to the aquatic species [13, 23, 24]. The phylogenetic placement of the aquatic species T. “raciborskii” is distinct from other aquatic species in the genus. It is placed among desert species and shares a common ancestor with several desert taxa. Reconstructing ancestral states allowed us to test different scenarios involving this aquatic species, including that T. “raciborskii” entered aquatic habitats secondarily or that the terrestrial species are independently evolved in this genus. Inclusion of a second, seemingly independent aquatic taxon is a novel aspect of our study, as is the use of multiple congeneric taxa from distinct habitats [10, 20, 25].

Given the irregularity of precipitation in terrestrial habitats, we hypothesize that terrestrial, but not aquatic, Tetradesmus species tolerate desiccation in a vegetative state, and that desert species would be more tolerant than algae isolated from temperate soils. We tested this by evaluating the recovery of physiological activity upon rehydration after desiccation. The rate of desiccation and the final value of relative humidity (RH) impact the time required for recovery of desiccation-tolerant plants (e.g., [26]). Thus, our second hypothesis is that two variables, the rate and maximum intensity of desiccation, impact the extent of recovery of Tetradesmus. To test these hypotheses, we subjected six species of Tetradesmus (four terrestrial and two aquatic) to desiccation under three different scenarios (RH ~ 5% over 2–5 h, 65% over 8 h, 80% over 16 h) followed by rehydration. We estimated photochemical yield of Photosystem II (Φ_PSII) from measurements of chlorophyll fluorescence during the desiccation–rehydration cycle to characterize the level of cell physiological activity. To account for possible within-species variation, which has been demonstrated for chlorophytes [10] and streptophytes [27], we used multiple strains per species when possible.

Desiccation and osmotic stress cause permanent damage to the ultrastructure of desiccation-sensitive cells (e.g., [28]); however, reversible changes in ultrastructure are also known for desiccation-tolerant organisms [29–31]. These changes include decrease of the cytoplasm volume, following expansion or undulation of cell wall, and degradation of thylakoid membranes. Therefore, our third hypothesis is that cell ultrastructure will change in both aquatic and desert species after desiccation, and that the change is permanent in the aquatic alga, but reversible upon rehydration in the desert lineage. To test this hypothesis, we compared cell ultrastructure of a selected aquatic and a desert species in hydrated, desiccated, and rehydrated states using transmission electron microscopy. We also tested the integrity of the plasma membrane under osmotic stress caused by a sorbitol solution, which mimics desiccation, using a vital fluorescent dye and confocal laser scanning microscopy.

Materials and Methods

Study Organisms

The response to desiccation and rehydration was studied in 13 strains of Tetradesmus belonging to 6 species: aquatic T. obliquus, T. sp. (listed at CCAP as “T. raciborskii” strain CCAP 276/35), temperate soil species T. dissociatus, and desert algae T. deserticola, T. adustus, and T. bajacalifornicus. All algae were grown in liquid Clear & Hom KSM medium (Supplementary Table S1) under a 12:12 h light/dark cycle (photon flux density ~ 200 μmol m⁻² s⁻¹) at 22 °C. Mixing of the cultures was achieved by orbital shaking at 0.4 rad/s.

DNA Extraction, Amplification, and Sequencing

To confirm the phylogenetic placement of T. obliquus strain UTEX 72, we obtained the sequences of three DNA loci (tufA, rbcL, and ITS2) and analyzed them with data from the other available species. Cells from culture aliquots of the strain UTEX 72 were concentrated, frozen, and then mechanically disrupted. Genomic DNA was extracted using the ZymoBIOMICS DNA Miniprep Kit. The tufA gene was amplified with the primer pair tufAF–tufA.870r [32, 33]. For amplification of the rbcL gene, we used the primer pair M35–M650r [34]. The ITS region was amplified using the primer pair ITS1–ITS 4 [32, 35]. Standard PCR protocol was carried out with GoTaq Green Master Mix (Promega Corporation, Madison, WI, USA) according to manufacturer’s recommendations. Prior to sequencing, amplification products were purified with Exosap-IT Express (Life Technologies Corporation, Carlsbad, CA, USA). DNA sequencing was performed by Eurofins Scientific with the same pairs of primers used for amplification reactions. Consensus sequences of three genes were obtained from forward and reverse sequences using Geneious 10.2.2 (https://www.geneious.com) and deposited to NCBI with accession numbers MT270139 for tufA, MT270138 for rbcL, and MT270137 for the ITS 2 region.

Phylogenetic Analysis

A three-gene concatenated dataset used in the analysis included six Tetradesmus species, some with multiple strains, and selected taxa from related genera (Supplementary Table S2). ITS2 was aligned using sequences together with the secondary structure (inferred by homology prediction) using ITS2 database [36, 37]. Alignments and the files necessary to run the phylogenetic analysis are available at DRYAD (doi:10.5061/dryad.sqv9s4n1t).

Substitution model and parameter values for the phylogenetic analysis were selected with Partitionfinder2 [38] using algorithms greedy [39] and PhyML [40]. The Akaike Information Criterion (AIC, [41]) was used to select the best model. Bayesian Interference (BI) was carried out with MrBayes 3.2.6 [42] available on the CIPRES Science Gateway [43]. The concatenated dataset was partitioned by gene and by codon position (for protein-coding genes). The HKY + I, F81, and HKY + G models were chosen for the first, second, and third codons of tufA, respectively, GTR + G was applied to all three codons of rbcL, and SYM + G model was selected for ITS 2. The analysis included two separate MCMC runs, each composed of four chains. Each MCMC chain ran for 2,000,000 generations, sampling trees every 100 generations. Upon completion, the runs were compared using Tracer v. 1.7 [44] and the first 25% of generated trees were discarded as burn-in. A 65% majority-rule consensus topology and posterior probabilities were then calculated from the remaining trees.

Maximum likelihood (ML) analysis of the concatenated dataset was carried out with partitioning by genes. Model GYR + I + G was implemented with following parameters: nucleotide frequencies A = 0.30920532, C = 0.16801733, G = 0.21847705, T = 0.3043003; substitution rates: AC = 0.40628109, AG = 1.567514, AT = 2.0983752, CG = 0.60725568, CT = 5.5017757, GT = 1.000000; Pinvar = 0.56788907; Gamma shape = 0.86898379. The ML tree was produced from the 1000 bootstrap pseudo-replicates using 65% majority rule. ML analysis and the bootstrap were performed using PAUP* V4.0a [45].

To further examine our hypotheses of the evolutionary history of the habitat transitions in the focal Tetradesmus species, we carried out ancestral states reconstruction for habitat of origin, as well as a test of the joint ancestral states for habitat at nodes A, B, C in Fig. 1. Find the details of this analysis in the Supplementary Material.

Fig. 1. Phylogenetic tree of desert and aquatic Tetradesmus species based on BI of tufA, rbcL, and ITS 2 rDNA.

Numbers associated with the nodes indicate support values for BI and ML analysis, respectively. Strains used in the desiccation experiments are indicated with a dot (solid shading indicates strains from the first experiment and measured with a PAM2500, gray shading indicate strains used in a second experiment and measured with a Junior PAM). Habitats of origin (aquatic, temperate soils, or desert soil crusts) are indicated by the color of a bar (blue, brown, and orange, respectively). The pie charts at the nodes show the estimated marginal posterior probabilities of the aquatic (blue) or terrestrial (orange) states at key nodes (see Supplementary Material for more details).

Desiccation and Rehydration Procedures

Desiccation experiments were carried out using desiccation chambers described in [46] and illustrated in Fig. S1. Different levels of relative humidity (RH) in the chamber were achieved by adding one of three desiccants to the chambers: 100 g of CaSO₄ (W. A. Hammond DRIERITE Co. LTD, Xenia, OH, USA) achieving 5% RH, a solution of 33 g LiCl in 100 ml of dH₂O for 65% RH, and a saturated solution of KCl in dH₂O (100 ml) for 80% RH.

Algal cell suspensions (50 μl, approximately 150,000 cells corresponding to chlorophyll concentration 30–40 mg ml⁻¹) were placed onto glass fiber filters (Whatman, Maidstone, UK) in replicates of four. Three filters (each containing a total of 200 μl algal suspension) at a time were then positioned on a perforated metal grid inside a transparent desiccation chamber containing the appropriate desiccant. A data logger (PCEMSR145S-TH mini data logger; PCE Instruments, Meschede, Germany) was placed in the chamber to monitor the change in RH. The chamber was then closed with a tight lid and the seal was secured with parafilm. The light probe was attached to a benchtop stand such that it was held fixed outside of the desiccation chamber, 12 mm distance from the algal samples. Placing the chamber on the benchtop maintained the distance from the probe to the samples. To take measurements of each algal spot on the three filters in the chamber (12 sample spots total), the chamber was rotated manually.

This setup allowed us to take the measurements rapidly, so we could run desiccations under different conditions in two different boxes subsequently. Positioning the light probe outside of the sealed chamber allowed us to take measurement without opening the chamber (which would have dramatically changed the conditions of desiccation).

Measurements of ΔF/Fm′ of PSII (Φ_PSII) were taken every 10 min during the desiccation period in dim light of ~ 10 μmol photons m⁻² s ⁻¹ at 22 °C. T. obliquus, T. deserticola, T. adustus, and T. bajacalifornicus were measured using a PAM 2500 chlorophyll fluorometer (Heinz Walz GmbH, Effeltrich, Germany), PAM 2500 settings: measuring light intensity 3, measuring frequency low (MF-L.) 200 kHz, measuring frequency high (MF-H.) 20,000 kHz; saturation pulse intensity 6, saturation pulse width 300 ms; actinic light intensity 3, width 0 s. Strains of T. dissociatus and T. sp. “raciborskii" (CCAP 276/35) were acquired later and measured with a Junior PAM (Heinz Walz GmbH, Effeltrich, Germany) with the same settings as for the PAM 2500. Dehydration experiments involving these species, along with a previously measured aquatic and terrestrial strain, followed the same protocol.

The dehydration process was assumed complete when the mean of measured ΔF/Fm′ for the algae on all filters reached zero. Then the chambers were briefly opened to rehydrate the samples by adding 50 μl of the KSM growth medium to each replicate spot on the filter and to replace the desiccant in a chamber with 100 ml of tap water to achieve higher RH (~96%). Then the measurements were resumed at the same intervals, without opening the box again until the end of the experiment.

Transmission Electron Microscopy (TEM) of Desiccated and Rehydrated Samples

We used TEM to compare cell ultrastructure in three physiological states (hydrated, desiccated, rehydrated). For this assay, we selected two species: aquatic T. obliquus (UTEX 393) and desert T. deserticola (EM2-VF30). The cultures were grown under the same conditions as for the desiccation experiment. For the hydrated cells, TEM sample preparation was performed directly on the culture suspension. The rest of the cells were spread on the uncovered Petri dishes and placed in a sealed box; desiccation was achieved by using 100 g of CaSO₄ (W. A. Hammond DRIERITE Co. LTD, Xenia, OH, USA) as a drying agent. Following 24 h of desiccation, half of the desiccated samples were used directly for TEM preparation, and the rest were rehydrated for 1 h by adding water to the Petri dishes.

Samples for TEM were prepared following the protocol described in [31]. Cells were fixed in 2.5% glutaraldehyde in cacodylate buffer (pH 6.8) for 1.5 h, washed in cacodylate buffer, then postfixed in 1% osmium tetroxide solution in cacodylate buffer overnight at 4 °C. The samples were dehydrated in increasing graded ethanol solutions and embedded in Spurr’s resin (Sigma-Aldrich, St. Louis, MO, USA). Ultra-thin sections were prepared, then viewed using FEI Tecnai 12 G2 Spirit BioTWIN TEM microscope.

Confocal Laser Scanning Microscopy (CLSM) under Osmotic Stress and Recovery

To test whether the plasma membrane of cells is fragmented during osmotic stress, we subjected cell suspensions of one representative species from each habitat to 4 M sorbitol and used the vital fluorescent dye FilmTracer™ FM™ 1-43 (green biofilm cell stain; Invitrogen Ltd. Paisley, UK) following the protocol described in [6]. Two aliquots of each selected species (aquatic T. obliquus strain UTEX 72, temperate soil T. dissociatus, desert T. deserticola strain SNI-2) were subjected to the osmotic stress in 4 M sorbitol solution for 1 h (cells under osmotic stress), after which sorbitol was replaced with deionized water in one of the aliquots (rehydrated cells). Control samples served as hydrated samples. All samples were then exposed to 20 μM FM 1-43, prepared from 20 mM stock solution in deionized water for 30 min prior to examination with Nikon A1R Spectral confocal microscope (Nikon Inc, Tokyo, Japan). Samples were excited with the argon laser beam at 488 nm; emission was collected at 500–550 nm (false colored green) and at 575–625 nm (false colored red).

Statistical Data Analysis with R

Data analysis and visualization were carried out with R (raw data and full code are available at DRYAD, doi:10.5061/dryad.sqv9s4n1t). We first calculated mean effective photosynthetic yield values from four individual measurements of each strain at each time point, then applied hierarchical cluster analysis to assign the “hydrated,” “dehydrating/dehydrated,” and “rehydrated” physiological states to the measurements. The time of rehydration (corresponding to the “rehydrated” physiological state) was recorded during data collection.

To compare recovery across species and for all investigated strains for each desiccation mode (RH 5%, 65%, 80%), we calculated recovery indices as the ratio of the mean effective photosynthetic yield value 10 min or 12 h after rehydration to the control hydrated value. We then used hierarchical cluster analysis of Euclidean distances to compare the recovery indices among the strains of desert and aquatic algae. The number of clusters was validated using gap statistics.

Results

Phylogenetic Analysis

The tufA and rbcL sequences obtained from T. obliquus strain UTEX 72 were very similar to these of the strain UTEX 393 and the ITS2 sequence of these strains were identical (see Table S2 for accession numbers of sequences used in the analysis). Phylogenetic analyses of the concatenated data set showed that these two strains are grouped into the same clade with high support. Phylogenetic analyses conducted using BI and ML resulted in identical tree topologies (Fig. 1), which agrees with previously published phylogenies of Tetradesmus [24, 47, 48]. Species isolated from desert soil crusts do not form a single clade, and instead are dispersed across the tree, with at least one desert species (T. bajacalifornicus) having a strongly supported sister relationship to an aquatic species, T. “raciborskii” (Fig. 1). Reconstruction of ancestral states favored a terrestrial ancestor for the aquatic species T. “raciborskii” over other possible reconstructions (Fig. 1, Table S3, S4).

Desiccation and Rehydration

The effective quantum yield of photosystem II (Φ_PSII) of hydrated cells was similar in all species included in the present study (approximately 0.6). As mentioned, the desiccation/ rehydration treatments of the temperate soil alga T. dissociatus and the aquatic species T. “raciborskii” were carried out separately using a Junior PAM, which resulted in the higher yield values for the hydrated cells; however, the overall patterns displayed by these species agree with the data collected with the PAM 2500. To compare the Junior PAM data with the measurements taken with the PAM 2500, two strains (T. obliquus strain UTEX 393 and T. bajacalifornicus strain ZA 1-7) were included in both the PAM 2500 and the Junior PAM desiccation experiments. The reduction in Φ_PSII during dehydration was also similar in all species and across desiccation modes. As illustrated in the example trace presented in Fig. 2, Φ_PSII remained constant for a considerable amount of time (60–1000 min depending on the absorbance power of the desiccant), but then dropped to zero within 30–80 min following the initiation of desiccation. Here, we use Φ_PSII as a proxy for general cell activity, and thus the process of dehydration begins when the Φ_PSII value decreases. This point of dehydration onset was determined using cluster analysis based on Euclidian distances. The loss of Φ_PSII followed similar dynamics in desert and aquatic species of Tetradesmus, indicating that the algae are unable to prolong physiological activity under desiccation stress.

Fig. 2. Example plot of the dehydration/rehydration cycle.

In black (left y-axis): representative example of raw measurements of PSII effective yield of a desert alga (T. adustus, strain LG2-VF29) taken every 10 min during dehydration at 65% RH, and rehydration. Data points correspond to the mean of four Φ_PSII measurements, error bars indicate 1 SD. In gray (right y-axis) is RH recorded during desiccation and rehydration. A rapid decrease in humidity coinciding with the shift from desiccation to rehydration was by the opening of the desiccation chamber to replace the desiccant with water and add liquid medium to each algal spot for rehydration

A striking difference among the aquatic and terrestrial Tetradesmus is seen upon rehydration, as illustrated in Fig. 3a. Algae of terrestrial, and especially desert, origin restored their photosynthetic capacity after dehydration (full recovery took 30–180 min). By contrast, the aquatic species showed no recovery of Φ_PSII even after 12 h of rehydration (with one exception, as discussed below). The response to rehydration also varied depending on the intensity of dehydration (Fig. 3b–d, Fig. S3). Rapid desiccation to 5% RH (over 2–3 h) caused the most severe damage to all species. Initially upon rehydration, desert species exhibited a short-term recovery of the photosynthetic activity but did not recover permanently. Less severe dehydration to 65% RH (8 h) and 80% RH (16 h) allowed a long-term recovery after rehydration of all terrestrial species (Fig. 3b–d, Figs. S2 and S3); however, the extent of recovery of photosynthetic activity varied among strains as described below.

Fig. 3. Example responses of aquatic and terrestrial Tetradesmus strains to acycle of desiccation andrehydration, as indicated by measurements of Φ_PSII.

Each data point represents a mean value of Φ_PSII (n = 4) and 1 SD (gray error bars), for measurements taken every 10 min. The hydration state of cells is indicated using different colored spots. We used cluster analysis to differentiate among the hydrated and desiccating states. Timepoints marked as rehydration were those following rehydration of dried spots. Data collected with different instruments are necessarily separated with (a) from Junior Pam and (b–d) from PAM 2500. a Behavior of T. “raciborskii” and T. obliquus (aquatic species), T. dissociatus (temperate soil species), and T. bajacalifornicus (desert species) when dehydrated at 65% RH and rehydrated. b–d Responses of three strains to three levels of drying (RH 5%, 65%, and 80%) followed by rehydration: b T. deserticola, c T. obliquus, d T. “raciborskii”

Cluster analysis of rehydration indices, the ratio of a rehydrated value of Φ_PSII to the initial or maximum hydrated Φ_PSII, provides a summary of responses across treatments and species (Fig. 4). After 10 min of rehydration (Fig. 4a,c), the most striking difference was between the aquatic species (no photosynthetic activity with an exception of one aquatic strain) and desert species (high photosynthetic activity). All desert species belonged to the same cluster when desiccated at 5% RH (Fig. 4a). Initial recovery of the temperate soil species was higher than that of the aquatic species, but the cluster analysis grouped them together for the treatments with 5% and 65% RH (Fig. 4c). Under milder dehydration conditions (65% RH), T. bajacalifornicus exhibited Φ_PSII values similar to those in the hydrated state (Fig. 4a), the aquatic taxa did not recover, and T. deserticola and T. adustus demonstrated 45–75% of their maximum Φ_PSII. Under the mildest treatment (i.e., 80% RH, 16 h), two aquatic strains, T. obliquus (UTEX 72) and T. “raciborskii,” remained inactive, whereas T. obliquus (UTEX 393) recovered some photosynthetic activity (Fig. 4a, c). Among desert species, T. bajacalifornicus again exhibited higher levels of recovery, with separate strains of T. deserticola and T. adustus grouped in the same cluster. Recovery of the temperate terrestrial species T. dissociatus was as high as the desert alga T. bajacalifornicus in this treatment (Fig. 4c).

Fig. 4.

Hierarchical cluster analyses of recovery indices (ratio of a rehydrated value to the initial hydrated photosynthetic yield) of Tetradesmus across desiccation treatments demonstrate that the habitat of the species (aquatic or terrestrial) as well as the desiccation mode influence their ability to recover from desiccation. Data measured with different instruments are necessarily separated, with (a) and (b) from PAM 2500, and (c) and (d) from Junior Pam. a, c After 10 min of rehydration, terrestrial algae are able to re-initiate their photosynthetic activity rapidly upon rehydration, and aquatic algae do not, although there is variation among strains of most species. b, d After 12 h of rehydration, the difference in recovery from different dehydration modes became more apparent. Within-species variation in the aquatic taxon is clear under the gentlest desiccation at 80% RH. Cluster number on the x-axis represents distinct groups (identified in cluster analysis of Euclidean distances, verified with gap statistic). Symbol shape indicates the native habitat of each species (circles for aquatic, square for temperate soil, and triangles for desert), and symbol color indicates the individual species. Multiple symbols of the same color correspond to different strains of each species

After 12 h of rehydration (Fig. 4b, d), the effect of intense desiccation on Tetradesmus cells became more apparent, as neither aquatic nor the desert algae maintained photosynthetic activity under desiccation at 5% RH. Cluster structure of responses by the desert species to the medium-rate dehydration (Fig. 4b, 65% RH) was similar to that of the 10-min rehydration (T. deserticola and T. adustus were clustered together, all strains reached ~ 75% recovery; T. bajacalifornicus was separated with the highest 100% recovery). Tetradesmus dissociatus isolated from temperate soils lost its photosynthetic activity after 24 h of rehydration (Fig. 4d) under this treatment. Under the mildest dehydration at 80% RH, a single aquatic strain T. obliquus (UTEX 393) recovered 75% of its hydrated Φ_PSII level, whereas T. obliquus (UTEX 72) and T. “raciborskii” failed to recover even after 12 h rehydration (Fig. 4b, d).

Cells in Different Hydration States Do Not Show Differences by TEM

The cells of two investigated species (T. obliquus, UTEX 393 and T. deserticola, EM2-VF30) had a single cup-shaped chloroplast with a pyrenoid surrounded by several starch grains of different sizes. Typical for green algae, thylakoids were single or in stacks of three to four and were never in larger stacks, contrary to what can be seen in embryophytes. Golgi bodies were composed of six to eight cisternae with attached vesicles, and numerous mitochondria could be seen in the cytoplasm (Fig. 5a). Desiccated cells did not show striking differences in their ultrastructure compared to the controls. The protoplasm did not shrink, thylakoid membranes were intact, and cytoplasm was not denser than in fully hydrated cells. Accumulation of electron-dense globules, known as plastoglobules (50–100 nm), was found inside the chloroplast of both species mainly under desiccated and rehydrated conditions (Fig. 5). Cellular membranes of T. obliquus appeared to have more contrast but did not show noticeable damage (Fig. 5b). No definite changes in cell ultrastructure were noticed upon rehydration in either investigated species. Thylakoid membranes of both species appeared intact (Fig. 5). The cytoplasm of T. obliquus was denser compared to the hydrated cells, but the plastoglobules were smaller compared to the desiccated cells (Fig. 5). Some rehydrated cells of T. deserticola contained a larger number of small vacuoles, and the chloroplasts contained numerous plastoglobules (Fig. 5b).

Fig. 5. TEM photomicrographs of an aquatic and terrestrial Tetradesmus in hydrated, desiccated, and rehydrated states.

a Ultrastructure of the aquatic species Tetradesmus obliquus (UTEX 393). b Ultrastructure of the desert species T. deserticola (EM2-VF30). Arrows point at plastoglobules. G—Golgi body, Chl—chloroplast, M—mitochondrion, N—nucleus, P—pyrenoid

CLSM Visualized Differences in Membrane Integrity

The fluorescent vital stain FM 1-43 did not penetrate the plasma membrane of the aquatic T. obliquus in the hydrated state (Fig. 6a), whereas cells exposed to 4 M sorbitol showed clear plasma membrane damage and rehydrated cells contained inner membranes stained with FM 1-43, indicating a severely damaged plasma membrane (Fig. 6b, c). In contrast, FM 1-43 did not penetrate the plasma membranes of the terrestrial T. dissociatus and T. deserticola in either of the physiological states (Fig. 6d–f and g–i, respectively), indicating a preservation of the membrane integrity.

Fig. 6. Fluorescence photomicrographs of an aquatic and terrestrial Tetradesmus in hydrated state, under osmotic stress, and rehydrated.

Effect of osmotic stress by 4 M sorbitol on the cells of T. obliquus UTEX 72 (a–c), T. dissociatus SAG 5.95 (d–f), and T. deserticola SNI-2(h, i) visualized by CLSM and lipid-soluble fluorescent dye FM 1-43. a T. obliquus fully hydrated control cells, only plasma membrane was exposed to the dye. b T. obliquus cells under osmotic stress, arrow points to the fracture in the cell membrane. c T. obliquus rehydrated cells with the dye binding to the intracellular material, indicating the damage to the plasma membrane by desiccation. d T. dissociatus hydrated cells. e T. dissociatus under osmotic stress, no indication of membrane damage. f T. dissociatus rehydrated cells, the membrane preserved its integrity. g T. deserticola hydrated control cells. h T. deserticola under osmotic stress, no indication of membrane fracturing. i T. deserticola rehydrated, the membrane integrity was preserved. Chl—chloroplast, P—pyrenoid

Discussion

Phylogenetic Relationships and Habitat Transitions in Tetradesmus

Of the 25 known species in the green algal genus Tetradesmus, five were described from terrestrial habitats [13, 23, 24]. Strain UTEX 72 was confirmed as a strain of T. obliquus in the present study. As shown in our study (Fig. 1) and in [24], not all aquatic taxa are reconstructed as closest sister taxa, with T. “raciborskii” sharing a common ancestor with the desert species T. bajacalifornicus. The other aquatic taxa T. obliquus and T. distendus were further separated from the first group by intervening terrestrial taxa. The ancestral state reconstruction analyses provide positive, but not strong, evidence of a single origin of terrestrial species in the genus Tetradesmus (Fig. 1). These analyses also indicate that T. “raciborskii” likely had a terrestrial ancestry and thus an independent transition to the aquatic habitat. Even with the inferred terrestrial ancestry of T. “raciborskii,” we tested the hypothesis of distinct responses to desiccation and rehydration by the aquatic and the terrestrial congeners.

Habitat of Origin Is Predictive of Vegetative Desiccation Tolerance Exhibited by the Tetradesmus Species

Our current understanding of vegetative desiccation tolerance is primarily based on the research of desiccation tolerant land plants, also called resurrection plants, and their streptophyte algal relatives [6, 11], or studies of chlorophytes [10] and of isolated lichen photobionts [49]. Physiological responses to desiccation of desiccation-tolerant green algae and mosses appears to have much in common (e.g., [19, 50–54]), including rapid (within minutes after rehydration) recovery even from lengthy and severe desiccation events.

A study by Gray et al. (2007, [20]) examined responses to desiccation and rehydration in a wide range of aquatic and desert green algae. They showed dramatic differences in the behavior of species adapted to these habitats, and that only desert algae were capable of recovering their full photosynthetic capacity after being dry for a period of 1–30 days. Conditions under which desiccation and rehydration occured influence the response of the algae. For example, desiccation in the dark resulted in the recovery of more species [20]. Cardon et al. [25] examined desert soil crust algae in the Scenedesmaceae (the family including Tetradesmus), which recovered photosynthetic activity 3 min after rehydration and to a much higher degree than the aquatic species. Cardon et al. [25] also demonstrated variation in recovery among the desert algae. Species included in the present study belonged to multiple genera, whereas our experiment aimed to investigate closely related species.

We first tested the hypothesis that the investigated terrestrial species are capable of recovering from dehydration events compared to the aquatic species. All species studied here lost their photosynthetic capacity upon desiccation. Upon rehydration, desert algae showed an immediate recovery, with 50–80% of initial signal being recovered in 10–30 min, followed by a period of slower change in which the photosynthetic activity stabilized within 1–3 h. However, rapid and severe desiccation at 5% RH was damaging even for these species (after short-term recovery of the photosynthetic activity immediately upon rehydration, photosynthetic capacity invariably declined again over time). Similar responses characterize desiccation-tolerant mosses (e.g., [50]) and algae [20, 25, 52].

In our experiments, the degree of desiccation is correlated with the time it takes to reach the target RH (5%, 65%, 80%). The duration of water loss from hydrated samples ranged between 4 and 16 h, which may potentially be experienced by cells in fundamentally different ways. Slow desiccation potentially allows cells to detect the signal and activate desiccation-induced protective pathways, whereas to survive rapid desiccation they must have these protective compounds accumulated prior to desiccation. The difference between these processes is demonstrated by comparisons of desiccation-tolerant angiosperms and bryophytes. Desiccation-tolerant angiosperms can only survive slow desiccation and demonstrate activation of protective pathways during desiccation [11, 55, 56]. Desiccation-tolerant bryophytes, on the other hand, can recover after rapid desiccation events due to constitutive production of osmolytes and other protective compounds during the hydrated state [57]. Interestingly, accumulation of protective compounds may depend on the growth conditions and on the age of plants under stress. For example, differential expression analysis showed that older cells of streptophyte algae were less stressed by desiccation compared to younger ones [58].

Our measurements of Φ_PSII suggest that the rate of photosynthesis decrease is similar in all three desiccation modes, but that the different desiccation modes varied in the timing of the dehydration onset. In other words, the treatments differed in how long Φ_PSII remains stable at the beginning of the experiment following the initiation of the desiccation treatment. We suggest that the cells were initially surrounded by a liquid layer of culture medium, which is attached to the cells by surface tension forces. Cells start losing water only after this extracellular water film is absorbed by the desiccant.

The modes of desiccation (humidity level and duration to reach it) have an impact on the desiccation tolerance phenotype of desert Tetradesmus (Fig. 4). The strongest desiccation (5% RH, 4–5 h) inflicted apparent damage even to terrestrial algae. Despite a short partial recovery of Φ_PSII after 10 min of rehydration in terrestrial algae was observed, in all of the studied terrestrial strains the Φ_PSII subsequently dropped to zero. A short-term initial recovery of Φ_PSII with a subsequent decline of the signal was also reported for other green algae exposed to desiccation (e.g., [54]). A lack of capacity of desert algae to fully recover from this harsh treatment is likely related to the difference between our experimental design and what the cells experience in nature. Desert soil crusts were shown to retain moisture for longer than the surrounding bare sand [17], which prolongs the hydration time and slows down desiccation. Although this treatment (5% RH, 4–5 h) likely was more extreme than what is experienced in nature, clear differences in response to desiccation are apparent in the aquatic and terrestrial species and indicate underlying physiological differences. Gentler modes of desiccation (RH 65 and 80% reached in 8 or 12 h, respectively) resulted in complete or almost complete recovery in all desert species. However, only one species (T. bajacalifornicus) recovered its maximum photosynthetic yield within 10 min of rehydration under both of these conditions. Among desert species, variation in recovery was higher for the strains of T. deserticola and T. adustus. Some fully recovered within 10 min when dehydrated at 80% RH, while others required more time to recover (Fig. S3). Dehydration of T. deserticola and T. adustus at 65% RH resulted in recovery of 45–75% of the maximum in the first 10 min of rehydration, and did not reach the initial maximum even after 12 h. After dehydration at 80% RH T. dissociatus behaved as the desert algae demonstrating recovery of 80% of the maximum photosynthetic yield after 10 min of rehydration. After dehydration at 65% RH the initial recovery of T. dissociatus was higher than that of the aquatic species (however, it clustered together with them), but no photosynthetic activity was recorded after rehydration for 12 h. This demonstrates that the dehydration tolerance phenotype of T. dissociatus is intermediate between the aquatic and desert congeners, making this species very interesting for future studies. Interestingly, the aquatic species T. “raciborskii” responded to desiccation and dehydration treatments the same way as another aquatic species T. obliquus (especially strain UTEX 72), despite its close phylogenetic placement with the desert Tetradesmus (Fig. 1).

The desiccation/rehydration profiles demonstrate variation in desiccation phenotype in this genus. The re-decline of photosynthetic activity after short-term recovery upon rehydration in the rapid and severe desiccation experiment shows the importance of extended data collection to comprehensively describe the response of a species to desiccation and rehydration. Variation in the desiccation phenotypes among the desert Tetradesmus, belonging to separate clades within the genus, may indicate differences in the mechanisms of vegetative desiccation tolerance in these species and opens the possibility for future development of the genus as a model group for investigations of the evolution of this complex trait.

To investigate variation among the strains of Tetradesmus species, we included multiple strains (2–4) for the species, where possible. In most of the cases, the within-species variation was minimal and different strains of a species clustered together (Fig. 4). The only exception was UTEX 393, a strain of the aquatic species T. obliquus, which recovered 75% of the photosynthetic activity after 12 h of rehydration following the mildest desiccation of our treatments (80% RH, 12 h). Such resistance to dehydration by an aquatic species is novel and was not known for Tetradesmus previously. Consistent recovery of UTEX 393 from dehydration suggests the presence of protective pathways even in the desiccation-sensitive strains. The genes responsible for these pathways are likely shared by all species of Tetradesmus and may prove key to discovery of the mechanisms of desiccation-tolerance in the desert species.

TEM Does Not Reveal Changes in Cell Ultrastructure during Osmotic Water Loss and Rehydration

Our observations of cell ultrastructure in two selected Tetradesmus species in different hydration states are drastically different from what can be found in the literature (e.g., [50, 59]). We did not detect cell plasmolysis (i.e., decrease in cell volume and retraction of the protoplast from the cell wall caused by a loss of water). Moreover, the cell wall retained the same thickness and none of its layers expanded. An undulation of the cell wall reported for Klebsormidiuim crenulatum [6] was not observed; slightly wrinkled cell walls were seen across all treatments and in both species, which may indicate an artifact of sample preparation. The plastoglobules detected in the chloroplast did not show distinct patterns when cells of each species were compared across different treatments and with the control. Despite the undisturbed ultrastructure of T. obliquus cells, they did not recover Φ_PSII after desiccation at 5% RH leading to the conclusion that the damage must have occurred not visible by structural changes by TEM.

Changes in Membrane Integrity Uncovered with CLSM

We tested the functional integrity of Tetradesmus cells using the amphiphilic styryl dye FM 1-43 as a vital stain. This stain binds to lipids and therefore will only be seen along the plasma membrane of intact cells, whereas in damaged cells with a fragmented plasma membrane, the membranes of the organelles will be stained as well. Cells of the aquatic T. obliquus had intact membranes only when hydrated (Fig. 6a). In cells under osmotic stress only the plasma membrane was stained, but a heterogeneous pattern of staining was found indicating plasma membrane alterations (Fig. 6b). The damage was more severe when the osmoticum was replaced with water: intense staining of intracellular material (Fig. 6c) suggests that this species is vulnerable to osmotic stress. By contrast, in cells of the temperate soil alga T. dissociatus and the desert alga T. deserticola, only the plasma membrane was stained (Fig. 6d–f and g–i, respectively) in all three physiological states (hydrated, under osmotic stress, rehydrated), indicating preservation of membrane integrity through the osmotic stress–relief cycle.

An absence of changes in cellular volume after desiccation or plasmolysis in Tetradesmus species may be related to the fact that these algae lack large vacuoles that are typical for many land plants and streptophyte green algae. In streptophytes, aquaporins located on the vacuolar and plasma membranes were shown to play a prominent role in desiccation tolerance (reviewed in [60]). The absence of a large vacuole may mean that even when fully hydrated, cells of Tetradesmus do not possess a storage of free water, which could be lost. In this case, the definition of desiccation tolerance as the ability to survive 80–90% water loss may not be applicable to these algae. Moreover, it is impossible to determine cell water content of hydrated Tetradesmus cells due to their microscopic size; water creates a film outside the cells which cannot be removed without risking dehydration of the cells. Thus, the method of subtracting dry plant mass from the hydrated mass that is commonly used for land plants (e.g., [19, 26]) cannot be used in unicellular algae. However, these algae exhibit the hallmarks of vegetative desiccation tolerance, as actively metabolizing cells can equilibrate with very dry air and recover from it upon rehydration.

Future Directions

Diverse species of algae are known to survive harsh conditions, and it is thought that survival from desiccation may be quite widespread across chlorophyte and streptophyte green algae (e.g., [20, 27, 31, 54]). In the present study, algae from a range of habitats were included, from aquatic to temperate soils to desert crust communities. As predicted, we found that a strain’s habitat of origin was associated with the ability of algae to recover from exposure to a variety of drying conditions in the vegetative stage, with aquatic taxa being the least able to cope with such water stress, desert taxa being most tolerant and the temperate soil species sharing some physiological responses of both desert and aquatic taxa. Recovery of one aquatic strain (T. obliquus, UTEX 393) at 80% RH reveals that some aquatic algae may tolerate dehydration stress to some extent. This ability may prove to be important for discovering evolutionary and molecular mechanisms of the emergence of desiccation tolerance in Tetradesmus. Aquatic Tetradesmus species are common in fresh water around the world (e.g., [61]), including regions where they experience seasonal or intermittent water restrictions, which should require an ability of cells to survive periodic environmental stresses, including desiccation. Many aquatic algae form tolerant dormant stages either as vegetative cells or as reproductive stages, such as zygotes (e.g., [62–64]). The genes imparting tolerance to these dormant stages may have been co-opted for vegetative desiccation tolerance by terrestrial Tetradesmus in a similar way that genes providing desiccation tolerance to seeds were co-opted in the vegetative tissues in desiccation-tolerant angiosperms (e.g., [65–67]). Future comparative omics studies of the closely related species of Tetradesmus from different habitats and having distinct desiccation tolerant phenotypes will enable investigations of the origins of the desiccation machinery in this group. And in particular, the inferred terrestrial ancestry of T. “raciborskii” can be further investigated by comparative analysis of its genomic and/ or transcriptomic characters with those of the closely related desert species (T. bajacalifornicus and T. adustus).

Supplementary Material

Supplementary material

The online version contains supplementary material available at https://doi.org/10.1007/s00248-020-01679-3.

Data Availability

New DNA sequence data are available in NCBI GenBank. Raw physiological data and scripts for data analysis were deposited to DRYAD (doi:10.5061/dryad.sqv9s4n1t).