From River to Desert: Using Chlorophyll Fluorescence to Test for Desiccation Tolerance Among

Hannah L. Gershone 1844 Blanchard Campus Center Mount Holyoke College 50 College Street, South Hadley MA 01075

Mentors: Dr. Zoe Cardon and Dr. Elena Peredo The Ecosystems Center 7 MBL Street Woods Hole, MA 02543

Semester in Environmental Science December 18, 2016

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Abstract Desert crusts are composed of a mix of organisms, including green algae, and provide many invaluable ecosystem functions. Desert crusts are a growing importance for ecosystem restoration. Understanding characteristics such as desiccation tolerance among crust organisms is critical for improving our restoration methods. I examined and compared the desiccation tolerance of three very closely related species of green algae from the genus : Scenedesmus deserticola, Scenedesmus bajacalifornicus, and . Scenedesmus deserticola was originally isolated from desert microbiotic crust. Scenedesmus bajacalifornicus was originally isolated from desert microbiotic crust, however, these cultures were lost. I used a Scenedesmus bajacalifornicus isolate from brackish water in a tidal river in Spain. Lastly, Scenedesmus obliquus is a freshwater isolate. To test for desiccation tolerance, I grew all of my algae under common garden conditions in liquid media. I then dried the algae slowly in the dark. After the algae were desiccated, I recorded fluorescence and humidity data during three cycles of desiccation and rehydration. I used the fluorescence data as an indicator of photosynthetic activity in the green algae. I found that S. obliquus most likely died during desiccation and/or rehydration. During the fluorescence assays, S. obliquus’ photosynthetic quantum yield of PSII was essentially zero. S. deserticola and S. bajacalifornicus had relatively high photosynthetic quantum yield of PSII values during all three hydration and desiccation cycles. However, S. bajacalifornicus had the highest average maximum photosynthetic quantum yield of PSII for all three cycles. I ultimately found S. obliquus had extremely low desiccation tolerance while S. deserticola and S. bajacalifornicus had high desiccation tolerance. Because S. bajacalifornicus had the highest average maximum photosynthetic quantum yield of PSII for all three cycles, it was the most desiccation tolerant.

Key Words Scenedesmus deserticola, Scenedesmus bajacalifornicus, Scenedesmus obliquus, green algae, desiccation, fluorescence, photosynthetic quantum yield of PSII, microbiotic crust, restoration

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Introduction Unappreciated for their physical beauty, microbiotic crusts are key components in desert ecosystems. Organisms combined with their by-products form crusts on top of the soil, creating a buffer against soil disturbance and erosion (Johnston 1997; USGS 2016). Crusts are invaluable to desert ecosystems; they can be the main carbon and nitrogen fixers in the ecosystem, store and filter water, and increase seed germination (Johnston 1997; USGS 2016). Large portions of crusts are composed of cyanobacteria, but a mix of diatoms, bacteria, rhizoid communities, fungi, and in particular, green algae are also found in crusts (Johnston 1997; Cardon et al. 2008; USGS 2016). I examined and compared the desiccation tolerance of three very closely related species of green algae from the genus Scenedesmus: Scenedesmus deserticola, Scenedesmus bajacalifornicus, and Scenedesmus obliquus. Two of these species made independent leaps to land (Cardon et al. 2008) and were isolated from desert crusts (Lewis and Flechtner 2004). One of the green algae species I studied that transitioned to land was Scenedesmus deserticola. S. deserticola (SNI-2) was originally isolated from desert microbiotic crust in San Nicolas Island, California (Lewis and Flechtner 2004). The second green algae species I examined, which also made an independent leap to land, was Scenedesmus bajacalifornicus. S. bajacalifornicus was first isolated from desert microbiotic crust in Baja, California and sequenced by Lewis and Flechtner (2004). However, the S. bajacalifornicus cultures isolated by Lewis and Flechtner (2004) were lost in Zoe Cardon and Elena Peredo’s lab. Instead, I tested another S. bajacalifornicus isolate (BEA 747B). BEA 747B was recently identified as S. bajacalifornicus, or a very similar species, based on a comparison of its 18S rRNA gene sequences to those of the S. bajacalifornicus desert isolates published by Lewis and Flechtner (2004). The S. bajacalifornicus isolate I tested is from brackish water in a tidal river in Spain (Nervión River Estuary-Station 3, Vizcaya, Banco Español de Algas 2016). The last green algae species I studied was S. obliquus (UTEX 393), which is a freshwater isolate (AlgaeBase 2016 B). Desiccation tolerance is critical in desert microbiotic crusts (Cardon et al. 2008). Desert crusts must survive in areas exposed to harsh sunlight where water availability is extremely low (Rosentreter and Belnap 2003). S. obliquus, the freshwater isolate, has very little desiccation tolerance (Cardon et al., unpublished data). On the other end of the spectrum is Scenedesmus deserticola, the desert microbiotic crust isolate, which is highly desiccation tolerant (Cardon et Gershone 3 al., unpublished data). Finally, Scenedesmus bajacalifornicus’ desiccation tolerance was unknown before my study. Both S. deserticola (desert) and S. bajacalifornicus (tidal river) undergo cycles of rehydration and desiccation in their respective ecosystems (Rosentreter and Belnap 2003). Desiccated algae undergo an additional stress; they’re still absorbing light. When an alga absorbs light energy, the energy first excites chlorophyll molecules in the antenna complexes (Amarnath et al. 2015). When an alga is hydrated, absorbed excitation energy from the antenna complexes is transferred to the plant’s photosynthesis reaction center where electrons flow into a ‘z scheme,’ eventually creating ATP and NADPH (Amarnath et al. 2015). A hydrated alga gives off huge chlorophyll fluorescence spikes, during very strong pulses of light that saturate photosynthesis, suggesting the alga’s photosynthetic machinery is active and functional (Veerman et al. 2007). However, when an alga is exposed to sustained high light intensity and/or begins to desiccate, the alga activates safety mechanisms (Gray et al. 2007; Veerman et al. 2007; Lunch et al. 2013). These safety mechanisms’ activities contribute to non-photochemical quenching, or NPQ (Maxwell and Johnson 2000; Veerman et al. 2007). When activated during desiccation, the safety mechanisms prevent light energy from being transferred to PSII (Veerman et al. 2007). We know these mechanisms are at work when we detect quenching of fluorescence from PSII at room temperature during saturating pulses of light (Veerman et al. 2007). If this dissipation didn’t occur, the photosynthetic machinery would be damaged in desert conditions! I hypothesized S. bajacalifornicus had a desiccation tolerance between S. deserticola and S. obliquus, but would be on the higher end of the desiccation range. This is because S. bajacalifornicus undergoes similar osmotic stress in a tidal river system as it does in a desert. S. bajacalifornicus must be able to handle salty conditions and long periods without water in both ecosystems. To test my desiccation hypothesis, I measured photosynthetic activity by collecting chlorophyll fluorescence data during cycles of desiccation and rehydration of the algae.

Methods

Algal Species and Culturing Methods: I grew cultures of all three green algae species: Scenedesmus obliquus, Scenedesmus deserticola, and Scenedesmus bajacalifornicus.

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Scenedesmus obliquus (UTEX 393): In the class (AlgaeBase 2016 B). S. obliquus was isolated from freshwater (AlgaeBase 2016 B) and obtained from the Culture Collection of Algae at the University of Texas at Austin (2016). They divide in groups of four and are shaped like crescents (personal observations). It has very little desiccation tolerance (Cardon et al., unpublished data).

Scenedesmus deserticola (SNI-2): In the class Chlorophyceae (Algaebase 2016 A). Originally isolated from desert soil surface in San Nicolas Island, California 33.2ºN latitude, 119.2º W longitude (Algaebase 2016 A; Lewis and Flechtner 2004) and sequenced by Lewis and Flechtner (2004). The cells are shaped like crescent moons, lemons, and/or bananas (Algaebase 2016 A; personal observations). S. deserticola is highly desiccation tolerant (Cardon et al., unpublished data).

Scenedesmus bajacalifornicus (BEA 747B): In the class Chlorophyceae (Banco Español de Algas 2016). It was collected from Vizcaya, Spain from the estuary of the Nervión River (43.36ºN latitude, -3.04º W longitude) by Aitor González and isolated by V. Cruz Alamo (Banco Español de Algas 2016). Its 18S rRNA gene sequence identified it as S. bajacalifornicus, based off of Lewis and Flechtner’s sequencing (2004). They are very round and shaped like bubbles (personal observations). Its desiccation tolerance is unknown.

I grew all three species of green algae under common garden conditions in a liquid culture mixture one half modified Bold Basal Medium (Stein 1973) and one half Woods Hole Medium (Stein 1973) as described in Lunch et al. (2013). I grew each species in a 250 mL Erlenmeyer flask. I filled every flask with 100 mL of the BBM and Woods Hole medium mixture and added 50 mL of already actively growing cultures of the respective algae. I grew the algae for one week in a Conviron CMP4030 growth chamber (Conviron, Winnipeg, Canada) at 25ºC with 12 hours of light (40 µE) and 12 hours of dark. All of the algae flasks were attached to a tube where air flowed in- cultures were ‘bubbled.’ Finally, I made algal stocks that grew on agar slants (1/2 BBM+1/2 Woods Hole, 1.5% agar). The algal stocks were kept in 20 mL screw- topped test tubes. Gershone 5

I took photographs of the algal cultures using an Olympus 13 x 40 Microscope (Sterling, VA) and a MU500-CK Amscope Microscope Digital Camera. I used Amscope 3.7 software on a computer to take photographs of the algae (Figure 1).

Desiccation Procedure: I poured 50 mL of my algae working solutions into 50mL Falcon Tubes (Corning) and allowed the algae to settle and concentrate by gravity. We then collected cells at the bottom of the tubes and transferred ~0.5 mL to 1.7 mL Eppendorf Tubes (Eppendorf). For each species, I pipetted six 40 µl concentrated dots of algal culture onto six clean and labeled cover glasses (18 mm by 18 mm, 1.5 mm thick, Fisher Scientific). The algae dots were slowly desiccated in a dark room using a custom drying apparatus. In order to control the humidity of air flowing into the chamber, a pump pushed air through an aerator in tepid water. The air, saturated with water vapor, then flowed through a tube to a condenser held at 0.5 degrees below room temperature. The condenser temperature was controlled by circulating water from an Isotemp 3016D water bath (Fisher Scientific). Therefore, air flowing out of the condenser was not completely, but slightly less, saturated at room air temperature. This moist air flowed into the drying chamber and desiccated the algae dots very slowly. The algae dots were kept in the desiccator for 17.5 hours, but dried within 10-12 hours. An astronomy camera (Acton PI 1 kb Versarray Cooled Camera, Princeton Instruments, Trenton, NJ) took images of the algae every half hour as the dots dried and changed shape from round and hydrated to flat and desiccated (Figure 2). The images were stored on a computer using WinView/32 software. Dried dots were stored in a tinfoil- covered box until they were assayed for fluorescence. This was to prevent algae from bleaching before fluorescence assays.

Cell Counts Using a Neubauer Haemocytometer (C.A. Hausser and Son, Philadelphia, P.A.), I calculated the average cell density for each of the settled, concentrated algae solutions used to make the 40 µl dots for the fluorescence assays. I did a 1:25 dilution using 2 µl of concentrated working solution and 48 µl of medium (modified 1/2 BBM+1/2 modified Woods Hole). I did two separate cell counts for each species and averaged the two to get average cell density.

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Fluorescence assay for desiccation tolerance: Once dried, I placed each cover slip with its respective dot of algae on it inside a plastic chamber with gas inlet and outlet ports connected to tubing. The cover slip became the bottom face of the chamber, and was held in place and sealed with nontoxic modeling clay. I monitored chlorophyll fluorescence from the algae dot through the cover slip with a flexible fiber optic guide was positioned ~1 mm from the cover slip. The fiber optic was connected to a Walz Junior-Pam chlorophyll fluorometer (Heinz Walz GmbH, Effeltrich, Germany). A constant stream of dry air flowed at 500 mL min-1 through the chamber over the algae, and a Licor 6262 infrared gas analyzer (Licor Inc., Lincoln, NE) measured the humidity of the air exiting the chamber. The humidity measurement showed the air moisture in the chamber, where the algae were, over time. An algae dot was initially dry when I placed it in the chamber. Then, I manually hydrated it by inserting a syringe needle through a chamber port and injecting water onto the desiccated algae. Air flowing through the chamber picked up water from the dot and slowly dried it. The Licor 6262 measurements reflect when I added water onto the algae dot and track the water evaporation dynamics from the dot as it dried. Algae underwent three cycles of desiccation and rehydration; the first injection of water was 10 µl during the first cycle and injections two and three were 3 µl each. Algae were constantly exposed to a measuring light of approximately 10 µmol photons m-2s-1 and were given a saturating pulse of 2,000 µmol photons m-2s-1 every three minutes. I measured fluorescence constantly through all of these cycles and the Junior PAM software saved my data. A Campbell Scientific CR10s datalogger (Campbell Scientific Inc., Logan, UT) recorded data from the Licor 6262. I did fluorescence assays for desiccation tolerance in triplicate for each species. I downloaded the fluorescence and humidity data into Microsoft Excel. I calculated photosynthetic quantum yield of PSII from the fluorescence data using Maxwell and Johnson’s (2000) yield equation:

�PSII = (F’m-Ft)/F’m

F’m = maximum fluorescence during a saturating pulse of light Ft = fluorescence before the pulse of light (steady state)

Results

Cell Counts Gershone 7

The average cell density of the settled, concentrated algae solutions used to make the 40 µl dots for the fluorescence assays was 3.2 x 108 cells mL-1 for Scenedesmus obliquus, 5.9 x 107 cells mL-1 for Scenedesmus deserticola, and 3.4 x 108 cells mL-1 for Scenedesmus bajacalifornicus (Table 1).

Fluorescence Assays of Desiccation Tolerance None of the three replicate dots within each species behaved exactly the same way. Acknowledging this, I’ll discuss fluorescence and Quantum Yield of PSII common trends across assays in the three replicate dots of each species, rather than stating anomalies within each algal dot. Variability in the number of saturating pulses during hydration and desiccation among Scenedesmus obliquus, Scenedesmus deserticola, and Scenedesmus bajacalifornicus was probably due to slightly different airflow rates through the chamber at the time.

Scenedesmus obliquus The humidity traces of Scenedesmus obliquus correlate with the fluorescence and traces, with one rising and falling with the other (Figures 3-5). For all three Scenedesmus obliquus trials, there was very little, if any, difference between maximal fluorescence (Fm’) and the steady state fluorescence (Ft) during the saturating pulses (Figures 3-5). S. obliquus showed no fluorescence spikes, even when hydrated, during the saturating pulses in all three cycles (Figures 3-5).

The photosynthetic quantum yield of PSII (�PSII) was essentially a flat line for all

Scenedesmus obliquus dots (Figures 3-5). When S. obliquus was completely desiccated, �PSII was 0 (Figures 3-5). Average maximum �PSII for the 10 µl injection was 0.095 (Figure 12). The species average maximum for both injections two and three (3 µl) was 0.043 (Figure 12). On average, maximum �PSII during the first injection was twice as high compared to maximum

�PSII during injections two and three (Figure 12), but overall �PSII during all injections was very low.

Scenedesmus deserticola

Fluorescence and �PSII rose with hydration and lowered with desiccation, creating three correlating curves seen for each Scenedesmus deserticola dot (Figures 6-8). All Scenedesmus deserticola dots, when hydrated, had a very large difference between maximal fluorescence Gershone 8

peaks (Fm’) and steady state fluorescence (Ft) (Figures 6-8). The difference between Fm’ and Ft decreases as Scenedesmus deserticola begins to desiccate (Figures 6-8). The decreasing difference is gradual in S. deserticola during desiccation, rather than abrupt, probably because of a slightly different airflow rate through the chamber. There appears to be two phases in increased

Ft after rehydration: a rapid increase followed by a gradual increase. In S. deserticola, the immediate rapid increase in Ft accounts for about three fourths (76%) of the total Ft increase during the 10 µl injection (Figures 6-8; Figure 13). The remaining increase in Ft is a slower process.

Because of the large difference between Fm’ and Ft, we see �PSII peaks during hydration and declines during desiccation (Figures 6-8). When S. deserticola was completely desiccated,

�PSII hovered around 0.1 (Figures 6-8). When S. deserticola was hydrated, �PSII immediately jumped to about 0.31 (10 µl injection) or a little under 0.31 (3 µl injection) (Figures 6-8; Figure

12). On average, injection 1 had an average maximum �PSII 0.03 greater than injection 2 and 0.05 greater than injection 3 (Figure 12).

Scenedesmus bajacalifornicus Just like S. obliquus and S. deserticola, the humidity traces of Scenedesmus bajacalifornicus correlate with the fluorescence and �PSII traces (Figures 9-11). Upon hydration, the difference between Fm’ and Ft is small, but continues to increase over time (Figures 9-11).

As with S. deserticola, increased Ft after rehydration appears to be biphasic. There’s a rapid Ft increase followed by a gradual Ft increase. The rapid increase in Ft accounts for only about one fourth (28%) of total increase in Ft during the 10 µl injection (Figures 9-11; Figure 13). Ft had a shorter time period to increase during injections 2 and 3 than during the 10µl injection (Figures

9-11). Thus, the biphasic Ft increase is still clear during injections 2 and 3, although less dramatic than during the 10 µl injection (Figures 9-11).

When S. bajacalifornicus was completely desiccated, �PSII hovered around 0.04 (Figures

9-11). When S. bajacalifornicus was hydrated, �PSII eventually climbed to an average maximum of 0.32 during injections 1 and 2 and 0.31 during injection 3 (Figure 12). In other words, average maximum �PSII was essentially the same during all injections.

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Discussion I used fluorescence assays to examine the desiccation tolerance of three closely related species of green algae: Scenedesmus obliquus, Scenedesmus deserticola, and Scenedesmus bajacalifornicus. As expected, the freshwater isolate, Scenedesmus obliquus, had the lowest desiccation tolerance among the algae. Each S. obliquus dot barely had any difference between maximum fluorescence (Fm’) and steady state fluorescence (Ft) during the saturating pulses, hydrated or desiccated (Figures 3-5). When S. obliquus was hydrated, it had extremely low Fm’ values, indicating its antenna complexes and photosynthetic machinery were damaged and unable to recover during desiccation (Figures 3-5). Although S. obliquus likely has safety mechanisms triggered when it’s under high stress, they were not equipped to handle desiccation for a prolonged period of time. S. obliquus most likely died during desiccation and/or rehydration, and the fluorescence emitted was from residual pigment. S. obliquus did have some variability within the species. S. obliquus Dot 5 had very small differences between Fm’ and Ft during the first cycle of hydration, but these extremely small differences were not present during the other two cycles (Figure 5).

Under complete desiccation, S. obliquus �PSII was 0, meaning no photosynthesis was occurring (Maxwell and Johnson 2000). Average maximum �PSII was highest during the first injection and lower for the other two cycles, suggesting that if there were any live cells present, they had a very low recovery rate (Figure 12). S. obliquus �PSII results are consistent with Gray et al.’s (2007) study of desiccation tolerance. Gray et al. (2007) found �PSII in aquatic algae still reflected signs of desiccation when hydrated after only 24 hours of desiccation. Scenedesmus deserticola had an extremely high desiccation tolerance. S. deserticola’s safety mechanisms disconnected antenna complexes from its photosynthetic apparatus (Zoe Cardon personal comment). These safety mechanisms prevented damage and caused low fluorescence yields when S. deserticola was desiccated. However, upon rehydration, we immediately saw a rapid increase in Ft after rehydration (Figures 6-8; Figure 13). The rapid increase was about three fourths of the total Ft increase during the 10 µl injection (Figures 6-8; Figure 13). Clearly, in S. deserticola the faster phase is a much larger part during the reconnection of the antenna complexes with the photosynthetic apparatus. The immediate rise in fluorescence and �PSII upon hydration indicates an incredibly fast recovery after desiccation for Gershone 10

17.5 hours (Figures 6-8). Csintalan et al. (1999) similarly found that highly desiccation tolerant mosses were able to regain pre-desiccation levels of photosynthesis almost immediately.

Scenedesmus deserticola �PSII during complete desiccation was never quite 0 (Figures 6-8).

Recovery time for �PSII increases with desiccation time (Gray et al. 2007), and if we had desiccated S. deserticola for weeks, it might not have reached its Fm’ peak so quickly.

Maximum average �PSII did slightly decrease during injections 2 and 3 in S. deserticola, relative to its value during injection 1 (Figure 12). This could indicate some damage to the photosynthetic apparatus as it endures three cycles of desiccation and rehydration. However, maximum average �PSII may also be a little lower during injections 2 and 3 because S. deserticola was hydrated for a third of the time it was in injection 1. Once S. deserticola began to desiccate, we saw slow quenching of fluorescence, as the difference between Fm’ and Ft became smaller and smaller (Figures 6-8).

Looking at average maximum �PSII, Scenedesmus bajacalifornicus appears to have the highest desiccation tolerance, with the greatest average maximum �PSII for all three injections

(Figure 12). Average maximum �PSII for S. bajacalifornicus remained essentially the same, from the first hydration and desiccation cycle to the last, indicating the alga is very tolerant of multiple desiccation and rehydration cycles (Figure 12). When completely desiccated, S. bajacalifornicus

�PSII is around 0.04, which is 0.06 less than S. deserticola when completely desiccated (Figures 6-11). This is probably due to slight differences in airflow rate at the time. The fluorescence curves may offer a reason behind why S. bajacalifornicus had the highest desiccation tolerance. The recovery of the photosynthetic activity appeared to have a rapid phase and a slow phase immediately after hydration. For S. deserticola, the rapid phase dominated, as it was three fourths of the overall recovery (Figures 6-8; Figure 13). In S. bajacalifornicus, the rapid increase in Ft during the first few seconds was only one quarter compared to the final Ft after 12-15 minutes of hydration (Figures 9-11; Figure 13). Overall, the slow, steady climb of Ft after rehydration indicates S. bajacalifornicus’ recovery rate of its photosynthetic activity was slower than S. deserticolas’ (Figures 6-11; Figure 13). Perhaps this slow climb is what helped S. bajacalifornicus to sustain a more recovered photosynthetic apparatus during all three desiccation and hydration cycles (Figure 12).

From the average maximum �PSII and initial Ft compared to overall Ft during hydration, we can conclude S. bajacalifornicus, while slow to increase in activity toward maximum �PSII Gershone 11 upon rehydration, is overall more desiccation tolerant. Scenedesmus obliquus, an aquatic green algae, doesn’t have the type of safety mechanisms, unlike Scenedesmus deserticola and Scenedesmus bajacalifornicus, to survive desiccation. Even when rehydrated, average maximum

�PSII barely rose above 0 for S. obliquus (Figures 3-5; Figure 12). Crusts are becoming more and more important for ecosystem restoration (Belnap and Eldridge 2003; Bowker 2007). One crust restoration method, called “inoculation,” introduces microbiotic crust organisms, taken from many different sites and/or cultured in a lab, to another site (Bowker 2007). While these crust mixes may grow well in lab, testing in field sites can be tricky with low success rates (Kubecková et al. 2003). By understanding the types of characteristics that allow crust organisms to thrive in particular environments, we can hone in on optimal crusts for various ecosystems, improving our restoration efforts. Finding the “right” crusts where plants are under osmotic stress would be a more efficient means than testing a wide variety of crusts that may not work- when crust recovery in disturbed ecosystems is already very slow (Belnap 1993; USGS 2016). S. bajacalifornicus, with its high desiccation tolerance and broad ecological tolerances, from rivers to deserts, could potentially thrive and be used in many different types of ecosystems.

Acknowledgements I would like to thank Zoe Cardon for her guidance, help, and support towards designing my project, laboratory work, analyzing data, and writing my report. Thank you to Elena Peredo for all her help during the designing and writing stages, and especially during the laboratory work. My project would not have been possible without either of them.

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AlgaeBase. 2016 B. Scenedesmus obliquus (Turpin) Kützing. Available from http://www.algaebase.org/search/species/detail/?species_id=27885. (accessed November 2016).

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Gray, D.W., L.A. Lewis, and Z.G. Cardon. 2007. Photosynthetic recovery following desiccation of desert green algae () and their aquatic relatives. Plant, Cell and Environment 30(1):1240-1255.

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Lewis L.A. and V.R. Flechtner. 2004. Cryptic species of Scenedesmus (Chlorophyta) from desert soil communities of western North America. Journal of Phycology 40(1):1127-1137.

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Figures and Tables

Table 1. Average cell counts of settled, concentrated algae solutions used to make the six 40µL dots for each species.

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A B C

D

Figure 1. A, Scenedesmus obliquus. B, Scenedesmus bajacalifornicus. C, Scenedesmus deserticola. D, Working solutions from left to right: S. obliquus, S. bajacalifornicus, and S. deserticola. A, B, and C taken at 400X with an Olympus 13 x 40 Microscope (Sterling, VA) and a MU500-CK Amscope Microscope Digital Camera. D taken using an iPhone 6 camera.

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Figure 2. 40 µl algae dots dried between 11:30 and 14:30 hours. Photos taken by an astronomy camera (Acton PI 1 kb Versarray Cooled Camera, Princeton Instruments, Trenton, NJ).

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S. obliquus Dot 1 Chlorophyll Fluorescence 700 600 500 Ft 400 Ft at pulse 7me 300 Fm' during pulse

Fluorescence (voltage) 200 100 0 0 10 20 30 40 50 60 70 80

Quantum Yield of PSII 0.4

0.3 Y (II) 0.2 Y (II)

0.1

0 0 10 20 30 40 50 60 70 80

Licor 6262 1 0.8 0.6 0.4 Licor 6262 0.2 0 0 10 20 30 40 50 60 70 80 Time (minutes)

Figure 3. When S. obliquus Dot 1 was hydrated, there was very little, if any, difference between maximal fluorescence and the steady state fluorescence. The quantum yield of PSII values were virtually 0; its antenna complexes and, subsequently, photosynthetic apparatus were damaged. The Licor 6262 humidity traces show 10 µl water was injected at ~11 minutes. The 3 µ l injections were given at ~41 minutes and ~57 minutes. Fluorescence and humidity traces were positively correlated; the three fluorescence curves occurred the same time as the three humidity ones. Fluorescence curves were most likely from residual pigment.

Gershone 18

S. obliquus Dot 3 700 Chlorophyll Fluorescence 600 500 Ft 400 Ft at pulse 7me 300 Fm' at pulse 7me 200 Fluorescence (voltage) 100 0 0 10 20 30 40 50 60 70 80

Quantum Yield of PSII 0.4

0.3 Y (II) 0.2 Y (II) 0.1

0 0 10 20 30 40 50 60 70 80

Licor 6262 1

0.8

0.6

0.4 Licor 6262 0.2

0 0 10 20 30 40 50 60 70 80 Time (minutes)

Figure 4. When S. obliquus Dot 3 was hydrated, there was very little, if any, difference between maximal fluorescence and the steady state fluorescence. The quantum yield of PSII values were virtually 0; its antenna complexes and, subsequently, photosynthetic apparatus were damaged. The Licor 6262 humidity traces show 10 µl water was injected at ~15 minutes. The 3 µ l injections were given at ~45 minutes and ~63 minutes. Fluorescence and humidity traces were positively correlated; the three fluorescence curves occurred the same time as the three humidity ones. Fluorescence curves were most likely from residual pigment.

Gershone 19

S. obliquus Dot 5 140 Chlorophyll Fluorescence

120 Ft 100 Ft at pulse =me 80 Fm' at pulse =me 60 40

Fluorescence (voltage ) 20 0 0 10 20 30 40 50 60 70 80 90

Quantum Yield of PSII 0.4

0.3 Y(II) 0.2 Y (II)

0.1

0 0 10 20 30 40 50 60 70 80 90

Licor 6262 1

0.8

0.6

0.4 Licor 6262 0.2

0 0 10 20 30 40 50 60 70 80 90 Time (minutes)

Figure 5. When S. obliquus Dot 5 was hydrated, there was very little, if any, difference between maximal fluorescence and the steady state fluorescence. Initial quantum yield of PSII values were high compared to S. obliquus Dots 1 and 3. However, quantum yield of PSII values eventually hovered around 0 or 0.05. S. obliquus Dot 5 antenna complexes and, subsequently, photosynthetic apparatus were clearly damaged. The Licor 6262 humidity traces show 10 µl water was injected at ~5 minutes. The 3 µl injections were given at ~47 minutes and ~68 minutes. Fluorescence and humidity traces were positively correlated; the three fluorescence curves occurred the same time as the three humidity ones. Fluorescence curves were most likely from residual pigment. Gershone 20

S. deser(cola Dot 2 Chlorophyll Fluorescence 700

600

500 Ft Ft at pulse 7me 400 Fm' at pulse 7me 300

200 Fluorescence (voltage )

100

0 0 10 20 30 40 50 60 70 80 90 100 Figure 13

Quantum Yield of PSII 0.4

0.3

0.2 Y(II) Y (II) 0.1

0 0 10 20 30 40 50 60 70 80 90 100

Licor 6262 1

0.8

0.6 Figure 15

0.4 Licor 6262 0.2

0 0 10 20 30 40 50 60 70 80 90 100 Time (minutes)

Figure 6. When S. deserticola Dot 2 was hydrated, there was a large difference between maximal fluorescence and the steady state fluorescence; antenna complexes were able to reconnect immediately with the photosynthetic apparatus upon hydration. Immediately after hydration, fluorescence first increased rapidly and then gradually until maximum fluorescence was reached. The quantum yield of PSII values were very high, reaching values just below 0.4; antenna complexes and photosynthetic apparatus suffered little damage. The Licor 6262 humidity traces show 10 µl water was injected at ~9 minutes after starting the assay. The 3 µl injections were given at ~45 minutes and ~69 minutes. Fluorescence, quantum yield of PSII, and humidity traces were positively correlated; the three curves for each occur during the same time. Gershone 21

S. deser(cola Dot 3 Chlorophyll Fluorescence 700 600

500 Ft 400 Ft at pulse 7me

300 Fm' at pulse 7me 200

Fluorescence (voltage ) 100 0 0 20 40 60 80 100 120

Quantum Yield of PSII 0.4 0.3 Y(II) 0.2 Y (II) 0.1 0 0 20 40 60 80 100 120

Licor 6262 1 0.8 0.6 0.4 Licor 6262 0.2 0 0 20 40 60 80 100 120 TIme (minutes)

Figure 7. When S. deserticola Dot 3 was hydrated, there was a large difference between maximal fluorescence and the steady state fluorescence; antenna complexes were able to reconnect immediately with photosynthetic apparatus upon hydration. Immediately after hydration, fluorescence first increased rapidly and then gradually until maximum fluorescence was reached. The quantum yield of PSII values were very high, reaching values around 0.3; antenna complexes and photosynthetic apparatus suffered little damage. The Licor 6262 humidity traces show 10 µl water was injected at ~10 minutes after starting the assay. The 3 µl injections were given at ~50 minutes and ~77 minutes. Fluorescence, quantum yield of PSII, and humidity traces were positively correlated; the three curves for each occur during the same time.

Gershone 22

S. deser(cola Dot 6 Chlorophyll Fluorescence 140

120

100 Ft 80 Ft at pulse 7me

60 Fm' at pulse 7me

40 Fluorescence (voltage ) 20

0 0 20 40 60 80 100 120

Quantum Yield of PSII 0.4

0.3

0.2

Y (II) Y(II)

0.1

0 0 20 40 60 80 100 120

Licor 6262 1

0.8

0.6

0.4 Licor 6262

0.2

0 0 20 40 60 80 100 120 Time (minutes)

Figure 8. When S. deserticola Dot 6 was hydrated, there was a large difference between maximal fluorescence and the steady state fluorescence; antenna complexes were able to reconnect immediately with photosynthetic apparatus upon hydration. Immediately after hydration, fluorescence first increased rapidly and then gradually until maximum fluorescence was reached. The quantum yield of PSII values were very high, reaching values around 0.3; antenna complexes and photosynthetic apparatus suffered little damage. The Licor 6262 humidity traces show 10 µl water was injected at ~11 minutes after starting the assay. The 3 µl injections were given at ~62 minutes and ~86 minutes. Fluorescence, quantum yield of PSII, and humidity traces were positively correlated; the three curves for each occur during the same time.

Gershone 23

S. bajacalifornicus Dot 1 Chlorophyll Fluorescence 700 Ft 600 500 Ft at pulse 7me 400 Fm' during pulse 300 200

Fluorescence (voltage ) 100 0 0 10 20 30 40 50 60

Quantum Yield of PSII 0.4 0.3 Y(II) 0.2 Y (II) 0.1 0 0 10 20 30 40 50 60

Licor 6262 1

0.8

0.6

0.4 Licor 6262 0.2

0 0 10 20 30 40 50 60 Time (minutes)

Figure 9. When S. bajacalifornicus Dot 1 was hydrated, there was a large, increasing difference between maximal fluorescence and the steady state fluorescence. Immediately after hydration, fluorescence first increased rapidly and then gradually until maximum fluorescence was reached. The quantum yield of PSII values were very high, reaching values around 0.35; antenna complexes and photosynthetic apparatus suffered little damage. The Licor 6262 humidity traces show 10 µl water was injected at ~6 minutes after starting the assay. The 3 µl injections were given at ~30 minutes and ~42 minutes. Fluorescence, quantum yield of PSII, and humidity traces were positively correlated; the three curves for each occur during the same time. Gershone 24

S. bajacalifornicus Dot 2 Chlorophyll Fluorescence 700

600 Ft

500 Ft at pulse 7me

400 Fm' at pulse 7me

300

200 Fluorescence (voltage ) 100

0 0 10 20 30 40 50 60 70 80

Quantum Yield of PSII 0.4

0.3 Y(II) 0.2 Y (II)

0.1

0 0 10 20 30 40 50 60 70 80

Licor 6262 1

0.8

0.6

0.4

Licor 6262 0.2

0 0 10 20 30 40 50 60 70 80 Time (minutes)

Figure 10. When S. bajacalifornicus Dot 2 was hydrated, there was a large, increasing difference between maximal fluorescence and the steady state fluorescence. Immediately after hydration, fluorescence first increased rapidly and then gradually until maximum fluorescence was reached. The quantum yield of PSII values were very high, reaching values around 0.35; antenna complexes and photosynthetic apparatus suffered little damage. The Licor 6262 humidity traces show 10 µl water was injected at ~12 minutes after starting the assay. The 3 µl injections were given at ~40 minutes and ~58 minutes. Fluorescence, quantum yield of PSII, and humidity traces were positively correlated; the three curves for each occur during the same time. Gershone 25

S. bajacalifornicus Dot 3 700 Chlorophyll Fluorescence 600

500 Ft Ft at pulse 7me 400 Fm' at pulse 7me 300

200

Fluorescence (voltage ) 100

0 0 10 20 30 40 50 60 70

Quantum Yield of PSII 0.4

0.3 Y (II)

Y (II) 0.2

0.1

0 0 10 20 30 40 50 60 70

Licor 6262 1

0.8

0.6

0.4 Licor 6262 0.2

0 0 10 20 30 40 50 60 70 Time (minutes)

Figure 11. When S. bajacalifornicus Dot 3 was hydrated, there was a large, increasing difference between maximal fluorescence and the steady state fluorescence. Immediately after hydration, fluorescence first increased rapidly and then gradually until maximum fluorescence was reached. The quantum yield of PSII values were very high, reaching values around 0.3; antenna complexes and photosynthetic apparatus suffered little damage. The Licor 6262 humidity traces show 10 µl water was injected at ~4 minutes after starting the assay. The 3 µl injections were given at ~31 minutes and ~49 minutes. Fluorescence, quantum yield of PSII, and humidity traces were positively correlated; the three curves for each occur during the same time. Gershone 26

Average Maximum Quantum Yield of PSII

10 µl 10 µl 3 µl 3 µl 3 µl 3 µl 0.4

b b 0.3 10 µl

Injec.on 1 0.2

Y (II) 3 µl 3 µl Injec.on 2 a 0.1 Injec.on 3 Anova p<0.05 Tukey's pairwise 0.0 S. obliquus S. deser.cola S. bajacalifornicus Species Figure 12. S. obliquus is statistically different than S. deserticola and S. bajacalifornicus. S. bajacalifornicus has the highest average maximum quantum yield of PSII for injections 1-3, indicating it has the highest desiccation tolerance among all three species.

Gershone 27

Average Ra#o of Ini#al Fluorescence Increase to Total Fluorescence Increase A;er Injec#on 1 10µl

1.0

0.8

10µl 0.6

Ra#o 0.4

0.2

0.0 S. bajacalifornicus S. deser2cola Species

Figure 13. After the first injection of water (10µl), the average initial rapid increase in fluorescence is only a little over one fourth (28%) of total fluorescence increase for S. bajacalifornicus. In S. deserticola, average initial rapid increase in fluorescence is about three fourths (76%) total fluorescence increase. S. obliquus was not examined for this figure because its low quantum yield of PSII indicated death sometime during desiccation or rehydration.