Strains used in this study and the phylogenetic position of Chromochloris
All strains assigned as Chromochloris zofingiensis available in public culture collections were investigated (Table S1; Supplemental Material). The genome of strain SAG 211-14 was published by Roth et al. (2017). This strain was originally sent in 2012 to the authors and sent back to SAG in 2017. We kept this strain (called here Genome) separately from the maintained strain of the collection for molecular comparison.
To test the monophyly of Chromochloris, the SSU and ITS rDNA of additional strains were sequenced (for details about origin, see Table S1). The DNA extraction, PCR, PCR purification, and sequencing were conducted with the methods described in Darienko et al. (2016). The selected strains are close relatives to Chromochloris as demonstrated by Fučíková and Lewis (2012). The SSU and ITS-2 rDNA sequences were aligned according to their secondary structures. The ITS-1 sequences were excluded from the data set because of the extreme length variations among the investigated strains. The phylogenetic analyses were inferred using the maximum likelihood, neighbor-joining, and maximum parsimony methods implemented in PAUP 4.0a167 (Swofford 2002). The best evolutionary model was calculated with the automated model selection tool in PAUP. The settings of the best model according to the Akaike information criterion is given in the legend of Fig. 1.
Genetic variability among the Chromochloris strains
Axenity test
All Chromochloris strains were cultivated on basal medium with beef extract (ESFl; medium 1a in Schlösser 1994). Before DNA extraction, all strains were tested and proven axenic. Four different organic agarized media were used for these tests: Trebouxia organic medium (TOM; Ettl and Gärtner 1995), malt peptone medium (medium 16 in Schlösser 1994), basal medium with peptone (ESP; medium 1b in Schlösser 1994), and ESFl medium. The plates were examined twice for contaminations in 2 and 4 weeks using a Stemi SV 11 binocular (Zeiss) and in addition checked with an Olympus BX60 (100× magnification) microscope using DIC-optics. Photographical documentation was achieved with a ProgRes C14 plus (Jenoptik) camera running ProgRes CapturePro V2.9.0.1 (Jenoptik). All cultures were cultivated at 20°C with the light:dark cycle of 16 h light and 8 h darkness at 50 μmol photons m-2 s-1 illumination.
DNA extraction for AFLP approaches
The extraction was performed from two-week-old cultures with an Invisorb Spin Plant Mini Kit (Stratec) in duplicates. Around 100 mg Chromochloris cultures (fresh weight) was placed into bead beating tubes and mixed with glass beads of 212–300 mm in diameter and shortly vortexed. The mixtures were mechanically disrupted using a PowerLyzer 24 (MoBio) for 10s at 3800 rpm. The further steps were done according to the protocols provided by the manufacturer. The quantity of DNA was assessed with a Qubit 3.0 Fluorometer (Thermo Fisher) and the quality was proven on 2% agarose gel stained with Midori Green. Samples containing long DNA strands (20-kb fragments) with a concentration of DNA between 20–25 ng µL-1 were used for further investigations.
AFLP protocol
We used the AFLP protocol provided by Müller et al. (2005) with small changes described below. Restriction and ligation reactions were combined in one reaction (Mannschreck et al. 2002). The combination of the restriction enzymes PstI-HF (cat.-No. R3140S) and EcoRI-HF (R3101S; all from New England Biolabs, Germany) was used in this study. Approximately 100–130 ng (5.5 μL) of high quality, non-shared DNA were incubated with 5.5 μL of following mastermix. The mastermix consisted of 0.23 μL H2O, 1.10 μL T4 DNA ligase buffer (10×; B0202S, New England Biolabs), 1.10 μL NaCl (0.5 M), 0.55 μL BSA (1 mg mL-1), 0.17 μL T4 DNA ligase (6 U μL-1; M0202S; New England Biolabs), 1 μL EcoRI adaptor (5 μM), 1 μL PstI adaptor (50 μM), 0.25 μL EcoRI-HF (20 U μL-1), and 0.1 μL PstI-HF (10 U μL-1). The mixture was incubated on a thermocycler for 2 h at 37°C followed by 1 h at room temperature. The success of the digestion was checked on a 1% agarose gel and then diluted 1:5 with sterile water.
Preselective PCR amplification
Two microliters of the diluted restriction-ligation mix was combined with a mastermix containing 7.25 μL H2O, 2.5 μL PCR buffer (5×; Bioline), 0.25 μL of each EcoRI+0 (20 μM) and PstI+0 (20 μM) primers, and 0.25 μL MyTaq Polymerase (5 U μL-1; Bioline). The mixture was briefly centrifuged at 1000 rpm and put into a thermocycler. The PCR reactions were performed with an initial denaturation of 5 min at 94°C, followed by 20 cycles of 20s at 94°C, 30s at 56°C, 120s at 72°C, and a final elongation of 10 min at 72°C. The samples were checked for quality on agarose gels and were diluted 10-fold for selective amplification with water.
Selective PCR amplification
1.5 μL of diluted samples from the previous step were combined with a 9.5 μL mastermix containing 2.25 μL H2O, 2.5 μL PCR buffer (5×), 3 μL EcoRI+A/C/G (mixture of three; 10 μM), 1.5 μL PstI+AC (100 μM), and 0.25 μL MyTaq Polymerase (5 U μL-1). The different EcoRI primers were labeled with 6-FAM, VIC, or NED fluorochromes (Applied Biosystems). The samples were briefly centrifuged at 1000 rpm and transferred into a thermocycler. The selective PCR reactions were performed with an initial denaturation of 5 min at 95°C, followed by 10 cycles of 20s at 95°C, 30s at 65°C, 120s at 72°C, followed by 20 cycles of 20s at 95°C, 30s at 56°C, 120s at 72°C, and a final elongation of 10 min at 72°C. Afterward, the samples were diluted 10-fold. Nine microliters of Hi-Di Formamide and 0.5 μL of GeneScan 600 LIZ size standard (cat. no. 4408399; Thermo Fisher) were pipetted to 0.5 μL of the diluted selective amplification products.
The complete AFLP reactions were performed twice from each DNA extraction at the same time.
MS-AFLP (Fig. 1)
The methylation-sensitive AFLP (MS-AFLP) with the two restriction enzyme combinations EcoRI/HpaII and EcoRI/MspI was performed using the same protocol described above. For the restriction/ligation, the enzymes HpaII (10 U μL-1; R0171S) and MspI (20 U μL-1; R0106S) with the MspI adaptor (5 μM) were used. The primers MspI+0 (20 μM) and MspI+C (100 μM) were used in the preselective and selective PCR amplifications, respectively.
AFLP and MS-AFLP fragment sizing and evaluation
Fluorochrome-labeled fragments were separated by capillary electrophoresis using an ABI Prism 3500 automatic sequencer (Applied Biosystems). AFLP fragments were evaluated in GeneMapper v5.0 (Thermo Fisher) and sized with GeneScan 600 LIZ size standard (Thermo Fisher). Fragments with a length of 50 bp to 600 bp were scored manually. The binary matrix (1 present, 0 absent) was constructed manually for AFLP (PstI). The scoring for MS-AFLP was also performed manually and the matrix contained four states (0 absent, 1 no methylation, both fragments HpaII and MspI present, 2 MspI present, HpaII absent, 3 MspI absent, HpaII present; Fig. 2). The methylation scoring was done according to Schulz et al. (2013).
For the detection of fragments, the “major rule” was applied for the four replicates. For both automated and manual evaluation, thresholds of 200 fluorescent units and the form of peaks were used. The differences between two different DNA extractions and restriction-ligation products were under 2% of number of total fragments.
Data analyses
The resulting matrices were exported into NEXUS format and further analyzed by PAUP 4.0a167 (Swofford 2002). The distance matrix of the binary matrix (AFLP-PstI) was calculated in PAUP using the neighbor-joining method and presented as cladogram. The support for specific nodes for the NJ tree was calculated with PAUP using the bootstrap method (Felsenstein 1985) with 1000 replicates (NJ and MP).
The descriptive statistics including the total number of fragments (Ftot), the percentage of polymorphic fragments (Fpoly%), and the numbers of unique fragments (Funi) were calculated as described in Schönswetter and Tribsch (2005).
Cryopreservation and fluorescein diacetate (FDA) vital staining
Before freezing, 750 μL algal culture were mixed with 750 μL of culture medium containing 10% DMSO, resulting in a final concentration of 5% DMSO as a cryoprotectant. Samples were frozen in an ICE Cube 14S Computer Controlled Rate Freezer (SY-LAB). The protocol was conducted according to Müller et al. (2007). The tubes containing the algae with cryoprotectant with a starting temperature of 20°C were frozen with a rate of one degree per minute to −40°C. Afterward, the tubes were incubated at this temperature for a further 30 min and then transferred into liquid nitrogen and put into the cryotank for storage.
Cryopreserved vials were completely thawed in a water bath at 41°C (around 3 min). Afterward, vials were sterilized with 70% ethanol, and all of the samples for one strain (0.5 mL of each replicate) were transferred into 9.5 mL of sterile BBM+V medium. The mixture was incubated in complete darkness for 24 h. For FDA staining, a mixture was prepared by dissolving 25 mg FDA crystals in a few drops of acetone. This mixture was filled up to 25 mL with methanol. One milliliter aliquots of incubated, thawed cultures were combined with 50 μL FDA solution (1% stock solution). The mixtures were incubated at room temperature for a minimum of 3 min. Afterward, samples were examined under an Olympus BX60 fluorescence microscope (WB filter). For the calculation of the survival rates, the SAG 211-14 (Genome) strain cultured under normal conditions was used as a reference. The culture contained 80% of stained cells. This value was used for correction during the calculation of survival rates according to the following formula:
$$ \mathrm{survival}\kern0.3em \left[\%\right]=100\times \frac{\#\mathrm{total}\kern0.3em \mathrm{cells}\kern0.3em \mathrm{postcryo}-\#\mathrm{dead}\kern0.3em \mathrm{cells}\kern0.3em \mathrm{postcryo}}{\#\mathrm{total}\kern0.3em \mathrm{cells}\kern0.3em \mathrm{postcryo}\ast 0.8} $$
Physiological experiments
All Chromochloris strains were pre-cultivated under standard culture conditions (20°C and a 16:8 h light:dark cycle) in Erlenmeyer flasks filled with 50 mL of Kuhl medium (medium 12 in Schlösser 1994). After 2 weeks of cultivation, the optical density (OD) of these cultures was measured photometrically at 567 nm with Genesys 20 (Thermo Scientific, MA). For cultivation in aerated culturing systems, these pre-cultures were diluted with Kuhl medium to the OD567 of approximately 0.06 to get a starting culture volume of 250 mL. These starting cultures were cultivated in a light thermostat (Kuhl and Lorenzen 1964) under the following conditions: 20°C, 16:8 h light:dark cycle, and 70–140 μmol photons m-2 s-1. The experiments were conducted under three different conditions: aeration with pressed air without enrichment (~0.04% CO2), enriched with 5% or with 10% CO2. Each treatment was done in triplicates. After the 9-day growth phase for biomass accumulation, the culture conditions were modified to induce carotenoid production in a stress phase of 7 days. The stress phase was induced by nitrogen depletion and an increase of light to 600 μmol photons m-2 s-1. For nitrogen depletion, the biomass grown after 9 days was collected by centrifugation, and the pellets were resuspended into the N-depleted Kuhl medium (with 1/10th nitrogen content of normal Kuhl medium). During the transfer to N-depleted medium, 20% of the biomass was separated from each sample for further analysis and storage, hence decreasing biomass prior to stress phase. These cultures were then transferred back to the light thermostat.
During the experiments, the contents of total carotenoids (Car), chlorophylls a (Chl a) and b (Chl b), as well as the biomass were measured on days 0, 2, 5, 7, 9, and 16.
Dry weight
Dry weight (DW) was directly measured on days 9 and 16 by freeze drying and indirectly proxied with OD correlations on days 0, 2, 5, and 7. For direct measurements, a 5 mL aliquot was taken from each sample, the OD at 567 nm was recorded, and then aliquots were placed into Eppendorf tubes. The samples were centrifuged and supernatant was discarded. The remaining pellets were frozen with liquid nitrogen briefly at −80°C and then freeze dried for 24–48 h. The DW was measured by subtracting the empty Eppendorf weight from the pellet plus Eppendorf weight and reported in gDW L-1.
Pigments
The pigment extraction (chlorophylls a and b, carotenoids) method and correlation equations were based on Lichtenthaler (1987). To measure the pigments on each day, a 1 mL aliquot was taken from the samples and centrifuged at 14000 × g for 30 min in a 2 mL tube, and the supernatant was removed. The pellets were mixed with glass beads. The samples were frozen in liquid nitrogen to enhance cell lysis. The cells were disrupted using a PowerLyzer 24 (MoBio) by 5000 rpm for 1 min. Lysed samples were centrifuged and cell debris partially pelleted (max rpm for 10s). Subsequently, samples were mixed with 1 mL acetone (100%) and incubated on thermomixer for 20 min at 70°C. After incubation, the samples were centrifuged (14000g × 20 min), and the supernatant was transferred to a spectrophotometer cuvette. The absorption at three wavelengths (473 nm, 650 nm, and 665 nm) was measured after blanking with acetone on the same Genesys 20 used for OD. The following equations were used to quantify pigment concentrations in mg μL-1 (see Lichtenthaler 1987):
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Chl a [mg L-1] = 11.24 E665 - 2.04 E650
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Chl b [mg L-1] = 20.13 E650 - 4.19 E665
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Total Chlorophyll [mg L-1] = Chl a + Chl b
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Total Carotenoids [mg L-1] = (1000 E473 - 1.90 Chl a - 63.14 Chl b) / 214
The cellular concentration of pigments in mg per gDW biomass was determined by dividing total Chl (mg L-1) or total Car (mg L-1) by dry weight (gDW L-1). Hereafter, total chlorophyll and total carotenoids will be referred to as Chl and Car. Statistical analysis was conducted using the Tukey test with 95% confidence intervals in Minitab 18, Minitab 18 Statistical Software (www.minitab.com).