Phylogeny and lipid profiles of snow-algae isolated from Norwegian red-snow microbiomes

Abstract

Snow algae blooms often form green or red coloured patches in melting alpine and polar snowfields worldwide, yet little is known about their biology, biogeography, and species diversity. We investigated eight isolates collected from red snow in northern Norway, using a combination of morphology, 18S rRNA gene and internal transcribed spacer 2 (ITS2) genetic markers. Phylogenetic and ITS2 rRNA secondary structure analyses assigned six isolates to the species Raphidonema nivale, Deuterostichococcus epilithicus, Chloromonas reticulata, and Xanthonema bristolianum. Two novel isolates belonging to the family Stichococcaceae (ARK-S05-19) and the genus Chloromonas (ARK-S08-19) were identified as potentially new species. In laboratory cultivation, differences in the growth rate and fatty acid profiles were observed between the strains. Chlorophyta were characterized by abundant C18:3n-3 fatty-acids with increases in C18:1n-9 in the stationary phase, whilst Xanthonema (Ochrophyta) was characterized by a large proportion of C20:5n-3, with increases in C16:1n-7 in the stationary phase. In a further experiment, lipid droplet formation was studied in C. reticulata at the single-cell level using imaging flow cytometry. Our study establishes new cultures of snow algae, reveals novel data on their biodiversity and biogeography, and provides an initial characterization of physiological traits that shape natural communities and their ecophysiological properties.

Keywords: 18S rRNA; ITS2 rRNA; fatty acids; imaging flow cytometry; microalgae; phylogeny.

Figure 1.

Sampling sites of macroscopically visible red-pigmented snow algae communities, Northern Norway. A 30 cm scale bar is shown (right).

Figure 2.

Microphotographs of eight algal isolates: R. nivale ARK-S01-19 (a1, a2, a3, a4, a5); R. nivale ARK-S02-19 (b1, b2); Stichococcaceae sp. ARK-S05-19 (c1, c2, c3); D. epilithicus ARK-S10-19 (d1, d2, d3); Chloromonas sp. ARK-S08-19 (e1, e2, e3, e4, e5, e6, e7, e8); C. reticulata ARK-S11-19 (f1, f2 f3, f4, f5, f6); C. reticulata ARK-S12-19 (g1, g2, g3, g4, g5, g6, g7, g8); X. bristolianum ARK-S13-19 (h1, h2, h3, h4, h5). Arrowheads indicate the papillae and arrows indicate contractile vacuoles. Scale bars are either 2 μm (with indication of the scale size near the bar) or 5 μm (no indication of the scale size). p = pyrenoid.

Figure 3.

ITS2 rRNA sequence-structure ML tree of Raphidonema with the new isolates (sequence) shown in bold. An asterisk indicates the type strain of the species R. nivale and the species names are as proposed by Yakimovich et al. (2021). The best model was JTT+G+I+F calculated by MEGAX 10.1.8. Numbers next to branches indicate statistical support values [ML bootstraps (1000 replicates)/Bayesian posterior probabilities]. The bootstrap support values above 70% and Bayesian posterior probabilities above 0.90 are shown. Thick lines indicate branches with full statistical support (ML/BI:100/1.00). Stichococcus bacillaris SAG 249.80 (MT078156) and D. epilithicus SAG 10.97 (MT078169) serve as the outgroup.

Figure 4.

ITS2 rRNA sequence and secondary structure-based ML phylogeny of Stichococcus-like species with the new isolates (sequences) shown in bold. The best model was WAG+G+F, calculated by MEGAX 10.1.8. The asterisk shows the type strain Deusterostichococcus epilithicus according to Pröschold and Darienko (2020). Numbers next to branches indicate statistical support values [ML bootstraps (1000 replicates)/Bayesian posterior probabilities]. The bootstrap support values above 70% and Bayesian posterior probabilities above 0.9 are shown. Thick lines indicate the branches with full statistical support (ML/BI:100/1.00). Pseudostichococcus monaillantoides SAG 379–4 (MT078184) and P. monaillantoides SAG 380–1 (KM020066) serve as the outgroup.

Figure 5.

The consensus structure of ITS2 rRNA sequence secondary structure between ARK-S05-19 (OM729986) and Prasiolales sp. S2RM26 (MK005091). Partial 5.8S and partial LSU stem regions of rRNA are included. One compensatory base change (CBC) between these two species within helix I is indicated in pink. Nucleotides highlighted in circles outside the yellow mark indicates a CBC between ARK-S05-19 (white circles) vs Prasiolales sp. S2RM26 (black circles). The conserved regions are indicated in blue with nucleotides (A, U, G, or C) and non-conserved nucleotides are shown in yellow. The ITS2 secondary structure and sequences were synchronously aligned and visualized using 4SALE.

Figure 6.

(A) 18S rRNA gene-based ML phylogenetic tree of Chloromonas. The asterisk shows the authentic strain of C. reticulata, the type species of the genus Chloromonas. Our new isolates (sequence) are shown in bold. Chloromonas clade 1,2 and 3 are delimited according to Hoham et al. (2002). (B) ITS2 rDNA ML phylogenetic tree of the Reticulata group showing the phylogenetic relationship of ARK-S11-19 and ARK-S12-19 with other C. reticulata. For both phylogenetic trees, the best model was K2+I calculated by MEGAX 10.1.8. Numbers next to branches indicate statistical support value [ML bootstraps (1000 replicates)/Bayesian posterior probabilities]. The bootstrap support values above 70% and Bayesian posterior probabilities above 0.90 are shown. Thick lines indicate the branches with full statistical support (ML/BI:100/1.00).

Figure 7.

A phylogenetic tree based on the alignment of 18S rRNA gene sequences using the ML method for Xanthonema, the new isolate (sequence) is shown in bold. The best model was TN93 + G +I calculated by MEGAX 10.1.8. Numbers next to branches indicate statistical support values [ML bootstraps (1000 replicates)/Bayesian posterior probabilities]. The bootstrap support values above 70% and Bayesian posterior probabilities above 0.9 are shown. Thick lines indicate the branches with full statistical support (ML/BI:100/1.00). Pylaiella littoralis CCAP 1330/3 (AY032606) and Nannochloropsis limnetica SAG 18.99 (AF251496) serve as the outgroup.

Figure 8.

Growth patterns of eight microalgal isolates at 2°C and 10°C (Experiment 1). The carrying capacity (maximum cell mass production, K, g·L⁻¹) and the intrinsic growth rate (r, d⁻¹) are shown in the gray box. Mean values (±standard error) of triplicates per isolate are given in the table. The lines show the predicted growth models at 2°C (blue dashed line) and at 10°C (grey line). The coloured points indicate the measured biomass concentrations over 20–45 days (n = 3), where lighter coloured points for each isolate indicates the measurements at 2°C. The asterisks indicate the mean values (± standard error) of duplicates per isolate due to an outlier that did not reach the stationary phase.

Figure 9.

Neutral lipid accumulation in C. reticulata ARK-S12-19 under nitrogen starvation (Experiment 3). (A) Analysis of TAG accumulation (bottom left) and the average TAG fatty-acid composition (top left) from n = 3 replicates in C. reticulata. 3t-16 : 1 indicates 3-trans-hexadecenoic acid. TAG content is determined by high performance thin-layer chromatography (HPTLC) together with standards (TAG; triacylglycerol, FFA; free fatty acids, DAG; diacylglycerol). The plate is labelled with Primuline and visualized under 254 nm UV light. (B) Flow-cytometric analysis of cell biovolume-normalized Bodipy^® fluorescence over 11 days (n = 3). The white vertical lines indicate the median of the frequency and the grey shaded points indicate individual cell data. Multispectral images show the accumulation of LDs in the cells. The Bodipy^® fluorescence (green) and chlorophyll autofluorescence (red) are shown. R, region: R2; round shaped cells, R4; oval shaped cells.