FEMS Microbiology Ecology, 92, 2016, fiw122
doi: 10.1093/femsec/fiw122
Advance Access Publication Date: 7 June 2016
Research Article
RESEARCH ARTICLE
Widespread green algae Chlorella and Stichococcus
exhibit polar-temperate and tropical-temperate
biogeography
Ladislav Hodač1,2,∗ , Christine Hallmann1 , Karolin Spitzer1 , Josef Elster3,4 ,
Fabian Faßhauer1 , Nicole Brinkmann5 , Daniela Lepka1 , Vaibhav Diwan1
and Thomas Friedl1
1
Experimental Phycology and Culture Collection of Algae (SAG), University of Göttingen, 37073 Göttingen,
Germany, 2 Department of Systematics, Biodiversity and Evolution of Plants (with Herbarium), University of
Göttingen, 37073 Göttingen, Germany, 3 Centre for Polar Ecology, University of South Bohemia, 37005 České
Budějovice, Czech Republic, 4 Institute of Botany, Phycology Centrum, Academy of Sciences of the Czech
Republic, 37982 Třeboň, Czech Republic and 5 Department of Forest Botany, University of Göttingen,
37077 Göttingen, Germany
∗
Corresponding author: Experimental Phycology and Culture Collection of Algae (SAG), University of Göttingen, Nikolausberger Weg 18, 37073 Göttingen,
Germany. Tel: +49(0)15115836127; E-mail: [email protected]
One sentence summary: Airborne unicellular microalgae are considered to have cosmopolitan distribution; however, molecular comparisons of
Chlorella- and Stichococcus-like species recognized lineages with either temperate-polar or temperate-tropical distribution patterns.
Editor: Rosa Margesin
ABSTRACT
Chlorella and Stichococcus are morphologically simple airborne microalgae, omnipresent in terrestrial and aquatic habitats.
The minute cell size and resistance against environmental stress facilitate their long-distance dispersal. However, the
actual distribution of Chlorella- and Stichococcus-like species has so far been inferred only from ambiguous
morphology-based evidence. Here we contribute a phylogenetic analysis of an expanded SSU and ITS2 rDNA sequence
dataset representing Chlorella- and Stichococcus-like species from terrestrial habitats of polar, temperate and tropical regions.
We aim to uncover biogeographical patterns at low taxonomic levels. We found that psychrotolerant strains of Chlorella and
Stichococcus are closely related with strains originating from the temperate zone. Species closely related to Chlorella vulgaris
and Muriella terrestris, and recovered from extreme terrestrial environments of polar regions and hot deserts, are
particularly widespread. Stichococcus strains from the temperate zone, with their closest relatives in the tropics, differ from
strains with the closest relatives being from the polar regions. Our data suggest that terrestrial Chlorella and Stichococcus
might be capable of intercontinental dispersal; however, their actual distributions exhibit biogeographical patterns.
Keywords: biogeography; green algae; Chlorella; Stichococcus; polar strains; SSU/ITS2
INTRODUCTION
Terrestrial species of Chlorella (Beijerinck 1893) and Stichococcus
(Nägeli 1849) are true survivalists among the green microalgae
(Chlorophyta). They inhabit biofilms covering natural and artificial subaerial substrates and dwell in soils (Carson and Brown
1976, 1978; Ettl and Gärtner 1995; Sharma et al. 2007; Rindi et al.
Received: 20 November 2015; Accepted: 27 April 2016
C FEMS 2016. All rights reserved. For permissions, please e-mail: [email protected]
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FEMS Microbiology Ecology, 2016, Vol. 92, No. 8
2009). Chlorella and Stichococcus have been reported by microscopical observations from nearly all soil types, including desert
soil crusts in Namibia (Büdel et al. 2009), humid tropical soils
in India (Ray and Thomas 2012), Central America (Archibald
1972) and Oceania (Arvik and Willson 1974; MacEntee, Bold and
Archibald 1977; Carson and Brown 1978) and polar desert soils
in Antarctica (Cavacini 2001; Fermani, Mataloni and Van de Vijver 2007) and the Arctic (Elster et al. 1999; Kaštovská et al.
2005, 2007; Patova and Dorokhova 2008). Langhans, Storm and
Schwabe (2009) recognized species of Chlorella and Stichococcus as
key players for monitoring the succession of biological soil crust
formation. Particular attention has been paid to the metabolic
facilities of psychrotolerant terrestrial strains of Chlorella vulgaris and Stichococcus bacillaris (Kvı́derová and Lukavský 2005;
Shukla, Kvı́derová and Elster 2011; Chen, He and Hu 2012; Hong
et al. 2015); both species include strains with the potential for
use in biotechnology (Lang et al. 2011; Olivieri et al. 2011, 2013;
Cadoret, Garnier and Saint-Jean 2012; Barreiro et al. 2013; Goiris
et al. 2014; Mudimu et al. 2014; Safi et al. 2014; Sivakumar, Jeong
and Lay 2014; Slocombe et al. 2015). The resistance against environmental stresses connected with metabolic versatility is a
common feature of polar microalgae, which have to cope with
low temperatures and shortages of nutrients and liquid water
(Elster 1999; Elster and Benson 2004; Kvı́derová and Lukavský
2005; Sharma et al. 2007). An extreme multistress resistance
was detected in a psychrotolerant cryptoendolithic strain of Stichococcus (isolated from Antarctica), which survived an experimental exposure to an Earth orbital space environment (Scalzi
et al. 2012).
Systematically, ‘Chlorella’ is the name for minute coccoid microalgae phylogenetically nested within the Chlorellaceae clade,
class Trebouxiophyceae (Huss et al. 2002; Krienitz et al. 2003;
Luo et al. 2010; Pröschold et al. 2010; Leliaert et al. 2012; Heeg
and Wolf 2015; Krienitz, Huss and Bock 2015). Whereas planktonic Chlorellaceae evolved into distinct forms, e.g. Micractinium
(Pröschold et al. 2010; Krienitz and Bock 2012) or Actinastrum (Luo
et al. 2010; Krienitz and Bock 2012), terrestrial members may
exhibit morphological convergence, characteristic for the ‘true’
Chlorella clade (Bock, Krienitz and Pröschold 2011) and for multiple Nannochloris-like clades (Henley et al. 2004). Morphologically
simple Chlorella- and Nannochloris-like species were repeatedly
recovered from Antarctica (Gilichinsky et al. 2007; Hu, Li and Xu
2008; De Wever et al. 2009).
‘Stichococcus’ can be as well encountered in the harsh terrestrial and freshwater environments of the Antarctic and Arctic (De Wever et al. 2009; Vishnivetskaya 2009; Khan et al.
2011). ‘Stichococcus’ is the common name for multiple rodshaped species phylogenetically nested within the Prasiola clade
(Handa et al. 2003; Neustupa, Eliáš and Šejnohová 2007; Novis,
Beer and Vallance 2008; Eliáš and Neustupa 2009). The Prasiola clade represents a trebouxiophyte lineage with the highest morphological diversity within the class (Leliaert et al. 2012)
and is widely distributed in freshwaters, marine and terrestrial ecosystems (Karsten et al. 2005; Rindi et al. 2007). Due
to their morphological simplicity, Stichococcus-like green algae
may veil a considerable phylogenetic diversity, which is still
in need of taxonomic revision (Neustupa, Eliáš and Šejnohová
2007; Sluiman and Guihal 2008; Karbovska and Kostikov 2012).
The rod-shaped Stichococcus species are scattered across the
whole Prasiola clade, but are not intermixed with morphologically distinct clades such as Pseudomarvania (Eliáš and Neustupa
2009) or the thallous Prasiolales (Handa et al. 2003; Rindi, McIvor
and Guiry 2004; Rindi et al. 2007). The Prasiola clade is clearly
nested within the core Trebouxiophyceae (Leliaert et al. 2012;
Lemieux, Otis and Turmel 2014; Turmel, Otis and Lemieux
2015), whereas plastid genome phylogenies place the Chlorellaceae outside the core Trebouxiophyceae (Lemieux, Otis and
Turmel 2014; Leliaert and Lopez-Bautista 2015; Turmel, Otis and
Lemieux 2015).
Chlorella, Stichococcus and a few other green algae of simple
morphology are often summarized as the so-called airborne algae (Sharma et al. 2007; Sharma and Rai 2010), which are supposedly ubiquitous in terrestrial ecosystems (Rindi et al. 2009, 2011).
The existence of cryptic algal species, which are morphologically
almost indistinguishable, was revealed by DNA sequence analyses (Boenigk et al. 2005; Rindi, Guiry and Lo 2008; Dal Grande
et al. 2014; Řı́dká et al. 2014; Škaloud et al. 2014, 2015; Ryšánek,
Hrčková and Škaloud 2015). Nevertheless, some species might
be cosmopolitans, as recently affirmed within some lineages
of terrestrial green microalgae, e.g. Asterochloris (Škaloud et al.
2015), Coccomyxa (Darienko et al. 2015), Diplosphaera (Fontaine
et al. 2012) and Klebsormidium (Ryšánek, Hrčková and Škaloud
2015; Ryšánek et al. 2016).
The remote polar regions provide an opportunity for investigating isolation by distance and speciation of microorganisms
(Martiny et al. 2006; Hahn et al. 2015). Pioneering molecular studies on algal diversity in Antarctica found genetic divergence between polar and non-polar species; some detected freshwater
Chlorella and Stichococcus species were supposed to be Antarctic endemics (Lawley et al. 2004; De Wever et al. 2009). Meanwhile the amount of sequenced algal strains and environmental
clones has dramatically increased, encouraging us to assemble
available data and check whether the Antarctic endemism might
be simply due to a sampling effect. We avoid the difficulty of the
ambiguous algal species concept (Leliaert et al. 2012, 2014); instead, we focus on monophyletic clades at low taxonomic level
(operational taxonomic units, OTUs) of identical or highly similar SSU sequences and review their geographical occurrences.
By focusing on common terrestrial and freshwater microalgae
Chlorella and Stichococcus, we aim to distinguish between clades
consisting of polar/temperate strains and clades consisting of
tropical/temperate strains.
MATERIALS AND METHODS
Data sampling and microscopy of the strains
We analyzed 118 new SSU and ITS2 rDNA sequences obtained
from the strains, isolates and clones listed in Table S1 (Supporting Information; Chlorella-like sequences, accession numbers
KX094751–KX094797, KX355545–KX355551, KX355553) and Table S2 (Supporting Information; Stichococcus-like sequences, accession numbers KX094798–KX094868, KX355552). The sequence
lengths varied as follows: (1355)1517-1619 bp (Chlorella SSU),
1598–1705 bp (Stichococcus SSU), 300–305 bp (Chlorella ITS2) and
268–381 bp (Stichococcus ITS2). The geographical origin of the new
polar, temperate and tropical sequences is shown in Fig. 1. Isolation technique to obtain algal strains from environmental material and DNA-sequencing procedure were described in Hodač
et al. (2015), while PCR cloning of the algal sequences from environmental samples was described in Hallmann et al. (2013). Morphological observations of the cultures were conducted using an
Olympus BX60 microscope (Tokyo, Japan) with Nomarski DIC optics and an attached ColorView III camera (Soft Imaging System,
Münster, Germany). Cell size measurements (diameter of spherical cells; length/width of elongated cells) were conducted in ImageJ (Schneider, Rasband and Eliceiri 2012) and were based on a
set of 50–100 cells per monoclonal culture.
Hodač et al.
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Figure 1. Origin of the newly sequenced Chlorella and Stichococcus strains. 1–7: Chlorella-like sequences (circles); 1: Canada (Ellesmere Island), 2: Svalbard, 3: Antarctica
(Killingbeck Island), 4: Antarctica (Adelaide Island), 5: Antarctica (King George Island), 6: Antarctica (Anchorage Island), 7: German Biodiversity Exploratories. 8–13:
Stichococcus-like sequences (squares); 8: German Biodiversity Exploratories, 9: Canada (New Scotia), 10: USA (Massachusetts), 11: USA (Alaska), 12: Ecuador (Podocarpus
National Park), 13: Indonesia (Bali, Lake Bratan). Detailed information on sampling sites is listed in Tables S1 and S2 (Supporting Information).
SSU rDNA phylogenetic analyses
The closest relative sequences to the new sequences were
searched for in GenBank using the BLAST algorithm (Altschul
et al. 1997). New and the GenBank SSU sequences were checked
for chimeras using Bellerophon (Huber, Faulkner and Hugenholtz 2004). Two separate SSU alignments were created, one for
Stichococcus-like sequences (Prasiola clade) and one for Chlorellalike sequences (Chlorellaceae). Both SSU sequence alignments
were computed using MAFFT v.6 (Katoh and Toh 2008) available
online. The aligned sequences were checked for possible misaligned positions in BioEdit 7.0.9.0 (Hall 1999). The SSU alignment, which included the new Stichococcus sequences and the
closest relatives from GenBank (Prasiola clade), contained 100
sequences/1709 positions (276 variable, 149 parsimony informative). Based on the AIC criterion in jModelTest 0.1.1 (Posada
2008), the GTR++I nucleotide substitution model was selected
as the best fitting of the Prasiola clade. A maximum-likelihood
phylogeny was computed in RAxML 7.0.4 (Stamatakis, Hoover
and Rougemont 2008) under the proposed model, and statistical support values were derived from rapid bootstrapping (1000
replicates) in the same program. An alternative maximumlikelihood tree search procedure, i.e. quartet puzzling, was applied for the same SSU alignment using Tree-Puzzle 5.2 (Strimmer and Von Haeseler 1996) available at the Mobyle-Pasteur
webserver (Néron et al. 2009). For this, the GTR+ substitution model was applied. To assess the approximate age of dichotomies within the Prasiola clade, relative node ages were
estimated using BEAST v1.8.2 (Drummond and Rambaut 2007;
Drummond et al. 2012) with 5000 000 steps (after 500 000 burnin) under the relaxed molecular clock option. Absolute ages were
estimated following (De Wever et al. 2009) by setting the minimum and maximum age of the Chlorophyta–Streptophyta split
at 700 and 1500 Ma. The maximum clade credibility tree was
visualized using FigTree (Rambaut 2007). The SSU alignment of
Chlorellaceae comprised 82 sequences/1668 positions (350 variable, 226 parsimony informative). The J2:G:5 substitution model
was proposed by Treefinder (Jobb, Von Haeseler and Strimmer
2004), which was subsequently used for maximum-likelihood
tree reconstruction. Confidence values were inferred from 1000
bootstrap replicates in the same program. The resulting tree was
visualized using MEGA6 (Tamura et al. 2013). For additional statistical support, Bayesian posterior probabilities were computed
in MrBayes 3.2.1 × 64 (Ronquist et al. 2012). We carried out two
MCMC runs for three million generations each with one cold
and three heated chains under the GTR++I evolutionary model
(parameters were estimated from the data); trees were sampled
every 100 generations. Additional statistical support values for
clades studied in detail were inferred from bioneighbor-joining
and maximum-parsimony methods as described in Hodač et al.
(2012). To summarize the genetic similarities within the alignments, the sequences were clustered into OTUs in the program
MOTHUR v.1.13.0 (Schloss et al. 2009). The gap positions were
excluded and two similarity thresholds were analyzed. Where
it was possible, the clades of highly similar (≥99.5%) SSU sequences were designed as OTU. The remaining sequences were
then clustered at lower similarity level (99.0%) and such clades
were designed as OTU∗ (marked with an asterisk). For additional
sequence comparisons, p-distances were computed in MEGA6
(Tamura et al. 2013).
ITS2 rDNA phylogeny and secondary structure analysis
First, 63 new ITS2 rDNA sequences were submitted to the BLAST
search (Altschul et al. 1997) in order to obtain their closest relatives for phylogenetic comparison. Subsequently, an online ITS2
database (Schultz et al. 2006; Selig et al. 2008; Koetschan et al.
2010, 2012) was used for ITS2 rRNA annotation (Keller et al.
2009). Minimum energy secondary structure models were computed from the annotated sequences using RNAstructure 5.3
(Reuter and Mathews 2010). For very closely related sequences,
a few were folded in RNA structure and the rest modeled using homology modeling in the ITS2 database (Wolf et al. 2005).
Ambiguous models were compared to those computed in the
mfold webserver (Zuker 2003). The ITS2 rRNA secondary structures were visualized by Varna 3.8 (Darty, Denise and Ponty
2009). Subsequently, two separate alignments of Chlorella vulgaris and Stichococcus-like sequences and structures were built
using 4SALE 1.7. (Seibel et al. 2006, 2008). In the same program,
compensatory base changes (CBCs) were computed between
pairs of sequences. The absence of CBCs might help to infer
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FEMS Microbiology Ecology, 2016, Vol. 92, No. 8
Figure 2. Microphotographs of polar strains of the Chlorellaceae. (a) Chlorella cf.
vulgaris L5 (Ø = 2.8–5.7 μm); (b) Chlorella sp.1 N9 (Ø = 2.9–5.4 μm); (c) Chlorella sp.2
L3 (Ø = 3.4–8.4 μm); (d) Muriella sp.2 L20 (Ø = 2.2–6.3 μm); (e) Marvania relative
L15 (Ø = 2.3–5.8 μm); (f) unidentified Chlorellaceae L24 (Ø = 2.6–7.3 μm). Scale
bar = 10 μm.
conspecificity in microalgae (Müller et al. 2007; Wolf et al. 2013).
The Chlorellaceae ITS2 alignment contained 39 sequences/321
positions (58 variable, 39 parsimony informative). A statistical
parsimony analysis was conducted with the very closely related ITS2 sequences of C. vulgaris and C. pituita using TCS v.1.21
(Clement, Posada and Crandall 2000) to generate ribotype networks. The connection limit was set to 95%. The Prasiola clade
ITS2 alignment contained 56 sequences/584 positions (252 variable, 189 parsimony informative). Based on sequence-structure
alignment, a neighbor-joining tree was computed in ProfDist
(Müller et al. 2004; Friedrich et al. 2005; Rahmann et al. 2006; Wolf
et al. 2008) using the GTR+ model and bootstrapping (1000 replicates). A splitting structure among closely related ITS2 ribotypes
was reconstructed by neighbor-net analysis in SplitsTree4 4.10
(Huson and Bryant 2006), applying uncorrected p-distances and
with ambiguities handled as averages. Bootstrap support values for internal splits were calculated with 1000 replicates. The
assignation of sequences into ribotypes was computed in DnaSP
v.5 (Librado and Rozas 2009).
RESULTS
Polar Chlorellaceae
The new Chlorella-like strains from polar and temperate regions exhibited two different gross morphologies: (1) Chlorellalike spherical to slightly elliptical cells (Ø = 2.8–8.4 μm) bearing one cup-shaped chloroplast with one (Fig. 2a) or two (Fig. 2b
and c) pyrenoids and (2) Muriella- or Nannochloris-like spherical
cells (Ø = 2.2–7.3) with a simple parietal chloroplast without a
pyrenoid (Fig. 2d–f). According to the SSU-based phylogenetic
analysis, all polar strains clustered close to sequences collected
from different geographical regions and using a ≥99.5% similarity threshold, we distinguished four different OTUs, which represented four different genera of Chlorellaceae (Fig. 3).
OTU1 (Chlorella vulgaris and relatives). The Antarctic strain
L4 (King George Island) and the Arctic strains L5 (Fig. 2a) and
L6 (Ellesmere Island) showed ≥99.5% SSU sequence similarity
to the authentic strain C. vulgaris SAG 211-11b. The SSU phylogeny suggested a divergence between the polar strains and
the remaining sequences within OTU1 which were mostly isolated from temperate regions (Fig. 3). We analyzed the observed
differences between the polar and temperate strains of C. vulgaris using the more variable ITS2 (Fig. 4), assigning them into
13 ribotypes (A, B, and E–O), which exhibited a total variability
of 54 polymorphic nucleotide positions (Fig. S1a, Supporting Information). The ribotypes A–K differed from C. vulgaris SAG 21111b by a few nucleotide polymorphisms. In contrast, ribotypes
L–O were more divergent from C. vulgaris SAG 211-11b and ribotypes A–K by sharing some identical nucleotide sites with C.
pituita ACOI 311 (Fig. S1a, Supporting Information). The ITS2 secondary structure model showed that a considerable amount of
nucleotide positions were conserved across all C. vulgaris ribotypes (including the polar ribotypes) and C. pituita (Fig. S1b, Supporting Information). Moreover, none of the ribotypes (not even
the polar ones) showed any CBCs against C. vulgaris SAG 21111b, but all showed at least three CBCs against C. pituita ACOI 311
(Fig. S2a, Supporting Information). No CBC was found between
the Antarctic strain L4 and the Arctic strain L6, although both sequences differed by 11 nucleotides in ITS2. Notably, the Antarctic
strain L4 (ribotype M) differed in only a single nucleotide from
the German isolate LH10HG2067 (ribotype L; Fig. S2b, Supporting Information). Otherwise, the same ribotypes were detected
from both aquatic and terrestrial habitats (i.e. ribotypes B, H, I;
Fig. S2b). Ribotype B was the most common ITS2 variety of C.
vulgaris in GenBank, including strains isolated from freshwaters
in Europe and USA (and probably also from China; Table S1, Supporting Information).
OTU2 (Chlorella spp.). The Arctic strain N9 (Fig. 2b) and the
Antarctic strain L3 (Fig. 2c) morphologically fitted into the
Chlorella clade sensu (Bock, Krienitz and Pröschold 2011). Both
strains clustered in a monophyletic clade without any named
strains, here delineated as OTU2, exhibiting ≥99.5% SSU similarity to other freshwater strains collected from the temperate
northern hemisphere and Antarctica. The SSU phylogeny suggested that OTU2 might be closely related to the genus Hindakia
C. Bock, Pröschold et Krienitz; however, the ITS2 phylogeny did
not support this close relationship (Fig. 4). The ITS2 inference
further suggested that strains N9 and L3 represented two different species, differing by 15 nucleotides and two CBCs. We detected a remarkably high ITS2 similarity between strain N9 from
the Arctic and strain KNUA034 (KM243327) from Antarctica, differing only by three nucleotide positions and one one-sided CBC
(=hCBC) in helix IV (Fig. S3a, Supporting Information).
OTU3 (Muriella terrestris clade). The Antarctic strains L20
(Fig. 2d) and N5 were nested within OTU3 (Fig. 3), containing
M. terrestris ASIB V38 and other terrestrial and aquatic strains
from temperate Europe and North America. The high SSU similarity (≥99.5%) between the Antarctic strains contrasted with
their genetic dissimilarity in the ITS2 marker (Fig. 4; Fig. S3b,
Supporting Information). The Antarctic strain L20 exhibited high
ITS2 similarity with the Antarctic clone Ant 8/104 and the
isolate LH08SG3009 from a German soil. The strains L20 and
LH08SG3009 differed by three nucleotides in ITS2 (Fig. S3b, Supporting Information).
OTU4 (Marvania relatives). The Antarctic strains L13, L15
(Fig. 2e), L23 and L32 showed high SSU similarity (≥99.5%) to
sequences collected from temperate regions (e.g. LH08SG3093;
Fig. 3). Marvania geminata SAG 12.88 and Nannochloris coccoides
CCAP 251/1b were the phylogenetically closest named relatives
of OTU4. The German strain LH08SG3093 exhibited a high ITS2
similarity to the Antarctic clone Ant 8/117 (JN653529), differing
by only 10 nucleotides (Fig. 4; Fig. S3c, Supporting Information).
In contrast to all the new polar strains, which were
assignable to known clades by their SSU sequences, strain
L24 from King George Island was an exception and represented a phylogenetically isolated branch within the Chlorellaceae (Fig. 3). Strain L24 (Fig. 2f) morphologically resembled
Hodač et al.
5
Figure 3. Maximum-likelihood tree of SSU sequences from polar Chlorella-like strains and other Chlorellaceae. Sequences are marked according to regions of origin:
blue = polar, green = tropical, gray = temperate, orange = hot desert. Authentic strains are marked by a ‘<’ sign. Numbers next to branches indicate statistical
support values (maximum-likelihood bootstraps/Bayesian posterior probabilities); clades of particular interest were additionally tested by maximum-parsimony and
bioneighbor-joining methods. OTU include sequences of ≥99.5% similarity. Drawings show morphology of selected strains (marked with an asterisk). Black circles
indicate which sequences originate from terrestrial (aerophytic/soil) and which from aquatic habitats (freshwater/marine).
minute Muriella- or Nannochloris-like species rather than the true
Chlorella.
Prasiola clade
The new strains of Stichococcus-like species exhibited the characteristic rod-shaped morphology (Fig. 5a–l) and varied in both
cell length (1.3–10.0 μm) and width (1.0–4.4 μm). Deviations from
the rod-shape morphotype, such as the Diplosphaera-like cell
packages, were observed in the isolate LH08SW1099 (Fig. 5j).
The Stichococcus strains did not substantially differ in chloroplast
shape and the detectability of pyrenoids using light microscope
was ambiguous. The Stichococcus-like SSU sequences originating
from polar, temperate and tropical regions (Table S2, Supporting Information) clustered into five clades of high inner similarity (≥99.5%; OTU1, OTU3-6) and four clades of lower inner
similarity (≥99.0%; OTU2∗ , OTU7∗ -OTU9∗ ; Fig. 6; Fig. S4; Table S3,
Supporting Information). Analyses of ITS2 polymorphisms
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FEMS Microbiology Ecology, 2016, Vol. 92, No. 8
Figure 4. Neighbor-joining tree of Chlorella-like species based on ITS2 sequences and secondary structures. Thick lines indicate bootstrap support values ≥80. CBCs
detected between sequences within a clade are given on branches as white numbers in black circles; gray numbers above CBCs give range of nucleotide differences
detected within clades. Sequences are marked according to regions of origin: blue = polar, green = tropical, gray = temperate; clade colors summarize the geographical
distributions (for detailed information see Table S1, Supporting Information; information in brackets is derived from the SSU data). Ant = Antarctic, Arc = Arctic, Eur
= Europe, N Am = North America. Clade denominations are adopted from the SSU phylogeny (Fig. 3).
(Fig. S5a, Supporting Information) and secondary structures
(Fig. S5b, Supporting Information) supported the monophyly of
most of the clades mentioned above, except for OTU2∗ , which
was non-monophyletic in the ITS2 inference and distinguished
as four different monophyletic (sub)clades (Fig. 7; Fig. S6, Supporting Information).
Polar Stichococcus
The Stichococcus-like SSU sequences originating from polar regions (Table S2, Supporting Information) were nested within
three different clades delimited as OTU2∗ , OTU8∗ and OTU9∗
(Fig. 6; Fig. S4, Supporting Information).
OTU2∗ (Stichococcus deasonii and relatives) included the authentic strain S. deasonii SAG 2139 (Alabama, USA) and other sequences originating mostly from terrestrial habitats (Fig. 6; Table S2, Supporting Information). The monophyly of OTU2∗ was
moderately supported in the SSU phylogenies (Fig. 6, Fig. S4,
Supporting Information) and partly rejected by ITS2 inference,
which recognized four monophyletic clades, i.e. S. deasonii relatives, Stichococcus clade2, Stichococcus clade6 and Stichococcus
clade7 (Fig. 7; Fig. S6, Supporting Information). Stichococcus
clade2 included one SSU sequence from the Arctic (EU282451;
Fig. 6) and two ITS2 sequences from Antarctica (e.g. HM490287),
differing from its European relatives (e.g. SAG 2482) by 6–9 nucleotide positions (Fig. 7). Another two SSU sequences of Antarctic strains clustered within OTU2∗ , i.e. S. bacillaris NJ-10 and S.
bacillaris s3; however, these sequences cannot be assigned to any
of the above-mentioned clades. The ITS2-supported Stichococcus clade6 and Stichococcus clade7, so far known only from the
temperate zone, might possibly be closely related to Stichococcus
clade2 (Fig. 7; Fig. S6, Supporting Information). The S. deasonii
clade of tropical species (e.g. clone KSK870SM6T; Fig. 7) clustered within SSU-based OTU2∗ ; however, the ITS2 phylogenies
suggested its isolated position within the Prasiola clade (Fig. 7;
Fig. S6, Supporting Information).
OTU8∗ (Diplosphaera) was a phylogenetically poorly resolved
clade consisting of multiple Stichococcus-like (S. chodati UTEX
Hodač et al.
7
Other Stichococcus clades
Figure 5. Microphotographs of the Stichococcus-like strains isolated from Germany and Ecuador. (a) Stichococcus clade2 LH08AW8104 (l = 4.0–10.0 μm; w =
1.8–3.2 μm); (b) Stichococcus clade7 LH10HG2063 (l = 4.5–8.7 μm; w = 1.8–2.8
μm); (c) Diplosphaera sp. LH08HW8075 (l = 3.2–6.2 μm, w = 1.9–4.4 μm); (d)
Diplosphaera sp. KS120SM6L (l = 1.3–4.9 μm, w = 1.9–3.4 μm); (e) Pseudostichococcus sp. LH08SW8044 (l = 3.4–9.8 μm; w = 1.6–3.3 μm); (f) Stichococcus clade1 SAG
2481 (l = 3.1–8.7 μm; w = 1.6–2.9 μm); (g) Stichococcus clade1 KS075SM6T (l = 2.9–
6.3 μm; w = 1.5–3.0 μm); (h) Stichococcus clade3 KS106CL6T (l = 2.5–5.0 μm; w =
1.5–2.4 μm); (i) Stichococcus clade4 SAG 2406 (l = 4.5–8.2 μm; w = 1.7–2.7 μm); (j)
Stichococcus clade5 LH08SW1099 (l = 3.0–6.3 μm; w = 1.0–4.2 μm); (k) Stichococcus
clade5 KS305SM6L (l = 3.3–6.8 μm; w = 1.4–3.2 μm); (l) S. jenerensis KS126SM6L
(l = 1.9–5.0 μm; w = 1.6–3.2 μm). Scale bar = 10 μm.
1177), Diplosphaera-like (Diplosphaera mucosa SAG 48.86) and
Chlorella-like (C. sphaerica SAG 11.88) species. The Diplosphaera
clade OTU8∗ was well supported in SSU phylogenies, yet without sufficient statistical support for finer inner resolution (Fig. 6;
Fig. S4, Supporting Information). The Antarctic strain D. mucosa SAG 48.86 was the only known polar species within
OTU8∗ . Other sequences originated from Central Europe (e.g.
LH08HW8075; Fig. 5c), the tropics (KS120SM6L; Fig. 5d), NorthAmerican deserts (e.g. S. chlorelloides BCP-CNP2-VF11B) and from
Tibet (clone QE28).
OTU9∗ (Pseudostichococcus) consisted of Stichococcus-like (S.
mirabilis CCAP 379/3, Pseudostichococcus monallantoides SAG 380–1)
and Desmococcus-like (Desmococcus spinocystis SAG 2067) species.
Both the SSU and ITS2 inference recognized the monophyletic
origin of OTU9∗ , yet with moderate statistical support. OTU9∗
included two strains isolated from Antarctica, i.e. Trebouxiophyceae sp. EO7-4 (freshwater) and S. minutus NJ-17 (terrestrial) and other strains collected from freshwater, marine
and terrestrial habitats of the temperate zone (e.g. Pseudostichococcus sp. LH08SW8044; Fig. 5e). The members of OTU9∗ exhibited high similarity in ITS2; for example, the freshwater
strains WB69 (Germany) and SAG 379-4 (North America) were
identical.
Six Stichococcus-like clades (OTU1, OTU3-6 and OTU7∗ ) included
sequences collected from temperate, tropical or both regions,
but lacked sequences from the polar regions.
OTU1 (Stichococcus clade1) consisted of terrestrial and aquatic
strains isolated from Germany (e.g. SAG 2481; Fig. 5f) and
Ecuador (KS075SM6T; Fig. 5g). The Ecuadorean strain differed
from the German strain KP09AW1004 by only 10 nucleotide positions within ITS2 and exhibited no CBCs (Fig. 7).
OTU3 (Stichococcus clade3) included European (Germany,
Swiss Alps), North-American (Yellowstone NP) and Ecuadorean
(Podocarpus NP) strains. The European freshwater strain SAG
2408 differed by three ITS2 nucleotide positions from the
Ecuadorean terrestrial isolate KS106CL6T (Fig. 5h).
OTU4 (Stichococcus clade4) consisted solely of sequences
of European provenance, including the freshwater strain SAG
2406 (Fig. 5i) and soil strain LH08SG1073, which differed by
12 nucleotides and one CBC in the ITS2. The Arctic strain
CCALA 906 (Svalbard; KF355941) had no closer relatives in GenBank than strain SAG 2406 (Germany), reaching 98.7% SSU
similarity.
OTU5 (Stichococcus clade5) contained terrestrial strains from
Europe (e.g. LH08SW1099; Fig. 5j) and Ecuador (e.g. KS305SM6L;
Fig. 5k). The Ecuadorean strain KS305SM6L differed from the European strain KP09AW1004 by four nucleotides in ITS2. OTU5
has been recorded also from Hawaii (environmental air sample),
since the SSU sequence KM462543 showed 99.82% similarity to
both European strains.
OTU6 (S. bacillaris and relatives) included predominantly
aquatic strains with the characteristic rod-shape morphology
(e.g. S. bacillaris SAG 379-1b) and strains morphologically assigned to Gloeotila (e.g. Gloeotila scopulina SAG 335–8). Other members of OTU6 originated from soil (e.g. clone HEW1B K3375), and
one sequence was obtained from the Southeast Pacific (strain
RCC 1054; KF899844). The ITS2 data suggested a close relationship between OTU6 and the Desmococcus clade (cell packagesforming/filamentous terrestrial microalgae), differing by only
two CBCs (Fig. 7).
OTU7∗ (S. jenerensis and relatives) was the only known
pantropic clade, containing the authentic strain S. jenerensis SAG
2138 (Southeast Asia) and the Ecuadorean strain KS126SM6L
(Fig. 5l), which differed by 13 nucleotides in the ITS2 (Fig. 7).
OTU7∗ further included sequences from terrestrial freshwater
and marine habitats of the temperate and tropical regions (Table S2, Supporting Information).
DISCUSSION
We summarized the phylogenetic diversity of new Chlorella- and
Stichococcus-like strains and environmental clones by clustering
their SSU sequences into OTU representing monophyletic clades
at a low taxonomic level. The SSU analyses confirmed that most
of the polar strains of Chlorella and Stichococcus exhibit ≥99.5%
similarity to strains from the temperate zone. Other temperate
strains, which exhibit ≥99.5% similarity to strains from the tropics, are phylogenetically divergent from their polar relatives. De
Wever et al. (2009) distinguished five different species of Antarctic Chlorellaceae and we recognized three of them as members
of our OTUs (Chlorella spp., Chlorella vulgaris and Marvania relative1). In accordance with Kochkina et al. (2014), we confirmed
the relatives of Muriella terrestris in Antarctica. For the first
time, we recorded Chlorella spp. and C. vulgaris relatives in the
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FEMS Microbiology Ecology, 2016, Vol. 92, No. 8
Figure 6. Maximum-likelihood tree of SSU sequences from Stichococcus-like species. Sequences are marked according to regions of origin: blue = polar, green = tropical,
gray = temperate, orange = hot desert. Authentic strains are marked by a ‘<’ sign. Numbers next to branches indicate statistical support values (maximum-likelihood
bootstraps/Bayesian posterior probabilities); clades of particular interest were additionally tested by maximum-parsimony and bioneighbor-joining methods. OTUs
include sequences of ≥99.5% similarity (=OTU) or ≥99.0% similarity (=OTU∗ ). Black circles indicate which sequences originate from terrestrial (aerophytic/soil) and
which from aquatic habitats (freshwater/marine); gray circles indicate missing information.
Hodač et al.
9
Figure 7. Neighbor-joining tree of Stichococcus-like species based on ITS2 sequences and secondary structures. Thick lines indicate bootstrap support values ≥80. CBCs
detected between sequences within a clade are given on branches as white numbers in black circles; the gray numbers above CBCs give range of nucleotide differences
detected within clades. Sequences are marked according to regions of origin: blue = polar, green = tropical, gray = temperate, orange = hot desert; clade colors
summarize the geographical distributions (for detailed information see Table S2, Supporting Information; information in brackets is derived from the SSU data). Ant =
Antarctic, Arc = Arctic, Eur = Europe, E Asia = East Asia, SE Asia = Southeast Asia, N Am = North America, S Am = South America. Clade denominations are adopted
from the SSU phylogeny (Fig. 6).
Arctic. The total phylogenetic diversity of Chlorellaceae in the
terrestrial habitats of polar regions is nearly as low as in other
arid regions, where <10 clades have been reported so far, e.g.
Chlorella sensu stricto, Micractinium and Meyerella (Lewis and
Lewis 2005; Flechtner, Pietrasiak and Lewis 2013; Fučı́ková, Lewis
and Lewis 2014). Only relatives of C. vulgaris and M. terrestris
have so far been detected in both extremely cold and hot
deserts.
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FEMS Microbiology Ecology, 2016, Vol. 92, No. 8
Polar Chlorellaceae with relatives in temperate
freshwaters and soils
Muriella terrestris and its closest relatives inhabit a variety of
habitats such as Central European and Antarctic soils (Shukla,
Kvı́derová and Elster 2011; Kochkina et al. 2014), Central European freshwater streams of high CO2 pressure (Hodač et al. 2015)
and North-American lakes (Fawley, Fawley and Buchheim 2004).
Remarkably, there is no molecular evidence of M. terrestris in
the Arctic regions. Other Chlorella-like OTUs analyzed here have
bipolar distribution and include members inhabiting both terrestrial and aquatic habitats. Marvania relatives were found in
permafrost soils in the Antarctic (Gilichinsky et al. 2007) and
Siberia (Vishnivetskaya 2009) and occur as well in Central European soils and North-American lakes (Fawley, Fawley and Buchheim 2004). The same is true for Chlorella spp., which are probably widely distributed across polar and temperate regions, as
documented by the multiple existing strains. Apart from the repeated evidence from Antarctic freshwaters and terrestrial habitats (Hu, Li and Xu 2008; De Wever et al. 2009; Hong et al. 2015) and
Svalbard soil (Shukla, Kvı́derová and Elster 2011), Chlorella spp.
occur as well in Central European and North-American freshwaters (Fawley, Fawley and Buchheim 2004; Hodač et al. 2015;
Park et al. 2015) and were recorded even from the South China
Sea (GU942201). Notably, freshwater strains of Chlorella spp. were
screened for application in biotechnology, e.g. CCAP 211/79 (Germond et al. 2013; Osundeko, Davies and Pittman 2013; Driver
et al. 2015). Similar attention was paid to the Antarctic psychrotolerant strains NJ-7 (Li et al. 2009; Lu et al. 2009, 2010) and
L5 (Shukla, Kvı́derová and Elster 2011), both close relatives of
the authentic strain C. vulgaris SAG 211-11b (the only authentic strain within the Chlorellaceae which is closely related to
polar species). Other terrestrial relatives of C. vulgaris SAG 21111b were recorded from North-American desert crusts (Flechtner, Pietrasiak and Lewis 2013) and German soils (this study).
Chlorella vulgaris and its relatives are among the best sampled
green microalgae available from GenBank. Chlorella vulgaris ITS2
sequences available from public databases and strains revealed
within this study represent a total of 13 different ribotypes (or
intragenomic copies) differing by 0–18 nucleotides. The closest
named relative of C. vulgaris SAG 211-11b is C. pituita ACOI 311
and both strains differ by 32 nucleotides (Fig. S1a, Supporting
Information) and four CBCs in ITS2 (Fig. S1b, Supporting Information). In another trebouxiophycean genus Coccomyxa, ITS2 ribotypes differing by up to 26 nucleotides were considered to be
conspecific (as proposed by Darienko et al. 2015, but questioned
by Malavasi et al. 2016). Therefore, we cannot exclude conspecificity of the C. vulgaris strains L4 (Antarctic soil) and L6 (Arctic
soil), which exhibit only 12 nucleotide differences in ITS2. The
same is true for the Chlorella spp. strains N9 (Arctic soil) and
KNUA034 (Antarctic freshwater), which differ by only three nucleotides. Widespread distribution of microbes, which are ecologically restricted to low-temperature environments, has been
documented, e.g. for cyanobacterial OTUs (Jungblut, Lovejoy and
Vincent 2010). However, in contrast to bacteria, which are more
restricted to a substrate type than eukaryotes (Fierer and Jackson 2006; Ragon et al. 2012), the Chlorellaceae may thrive in lowtemperature environments as well as in soils and freshwaters of
the temperate zone.
Another possible hint of environmental selection might be
the striking lack of the Chlorellaceae in the terrestrial habitats
of tropical rainforests. On the one hand, morphology-based surveys from Southeast-Asian rainforests could not recognize any
terrestrial ‘true’ Chlorella morphospecies (Neustupa and Škaloud
2008, 2010). On the other hand, terrestrial Chlorella-like microalgae described from tropical rainforests belong to lineages different from the Chlorellaceae, e.g. the Watanabea (Zhang et al.
2008; Neustupa et al. 2009; Suutari et al. 2010; Song et al. 2015) or
Jenufa clades (Němcová et al. 2011; Hodač et al. 2012). Moreover,
SSU cloning of microalgae from Ecuadorian rainforests (Podocarpus NP) revealed characteristic terrestrial taxa such as Stichococcus and Coccomyxa, but failed to detect any common terrestrial
Chlorellaceae (unpublished data of the coauthors F.F. and K.S.).
The only detected Chlorella sequence was a close relative of the
freshwater species C. sorokiniana, a well-known thermotolerant
microalga (de-Bashan et al. 2008; Zheng et al. 2013). Chlorella
sorokiniana was recorded from both cold and hot environments,
including Antarctic freshwater (De Wever et al. 2009) and NorthAmerican deserts (Flechtner, Pietrasiak and Lewis 2013). The
widespread occurrence of C. sorokiniana in humid tropics was
confirmed in Central America (de-Bashan et al. 2008), South
America (de-Bashan et al. 2008; Bashan et al. 2016) and Southeast Asia (Marimuthu 2011). The Chlorellaceae known from the
tropics are exclusively freshwater species, e.g. Hindakia fallax
(Kenya), C. pulchelloides (Mexico), C. rotunda (Angola), C. singularis (Kenya) and C. volutis (Kenya; Bock, Pröschold and Krienitz
2010; Bock, Krienitz and Pröschold 2011). As well desert Chlorellaceae have closest relatives in the temperate phytoplankton,
e.g. Meyerella (Fučı́ková, Lewis and Lewis 2014) and Micractinium
(Flechtner, Pietrasiak and Lewis 2013).
Polar Stichococcus with relatives in temperate
freshwaters, seas, soils and lichens
Similar to the polar Chlorellaceae, the polar strains of Stichococcus are nested within clades composed of closely related
sequences collected from temperate freshwaters and marine
samples. As a rule, phylogenetically different temperate Stichococcus sequences clustered together with their tropical relatives. A bipolar distribution was detected only in Stichococcus
clade2, composed of environmental clones from the McMurdo
Dry Valleys (Khan et al. 2011) and Stichococcus sp. 594-GA18, the
only strain known from the Arctic (Vishnivetskaya 2009) and isolated from a 4.65 m depth in Siberia. Pseudostichococcus is another
clade containing Antarctic strains (De Wever et al. 2009; Chen, He
and Hu 2012) and other strains isolated from temperate soils,
lichens (Thüs et al. 2011) and freshwaters (Hodač et al. 2015).
When compared to the terrestrial Chlorellaceae, which are
morphologically less varied, species of terrestrial Stichococcus exhibit striking cell-size variation (Fig. S4, Supporting Information) and unicellular versus cell-package stages (e.g. Stichococcus clade5; Fig. 5j). Terrestrial species of Stichococcus thrive in
a wider range of substrates, which might be an advantage in
competitive ecosystems like humid tropical rainforests. According to the molecular-clock phylogeny by De Wever et al. (2009),
Chlorellaceae colonized Antarctica before the Stichococcus-like
species appeared. The presence of deep-branching Chlorella-like
descendants in Antarctica suggests adaptation to cold and dry
environments. Our more detailed molecular-clock phylogeny
of Stichococcus, calibrated according to De Wever et al. (2009),
determined the first appearance of Stichococcus-like clades approximately during the Jurassic-Cretaceous (Fig. S7, Supporting Information). The Pseudostichococcus clade originated from
the earliest known split within the Prasiola/Stichococcus lineage and exhibits a polar-temperate distribution in the present.
The remaining Stichococcus clades might have originated in
more humid rather than dry desert environments, because
Hodač et al.
deep-branching clades like Stichococcus jenerensis and Stichococcus clade1 show distributions restricted to the tropics and
temperate regions.
Although we could not unambiguously confirm any case of
high similarity between polar and tropical sequences, we recognized one exception within the Diplosphaera clade. Diplosphaera
is the only Stichococcus-like clade, which includes desert members; the strains were collected from soil crusts in North America (Lewis and Flechtner 2002; Lewis and Lewis 2005; Flechtner, Pietrasiak and Lewis 2013) and Tibet (Wong and Lacap
2010). Other terrestrial members of the Diplosphaera clade were
recorded from the Ecuadorian rainforest (e.g. strain KS120SM6L)
and show striking similarity (only three different nucleotides in
ITS2) to the isolate LH08HW8075 from a forest soil in Germany.
Considering the SSU phylogenies, the isolate LH08HW8075 is a
close relative of the Antarctic strain Diplosphaera mucosa SAG
48.86. Diplosphaera spp. are also common lichen photobionts
(Thüs et al. 2011; Fontaine et al. 2012, 2013), but have also been
recorded from an acidic freshwater (Aguilera et al. 2007) and even
from an oceanic sediment (AB183601).
We conclude that Chlorella- and Stichococcus-like OTUs (at
low taxonomic level) are widespread, on one hand, and differ
in temperate-polar and temperate-tropical distributions, on the
other hand. Microorganisms can effectively disperse around the
globe via air (Herbold et al. 2014) and oceans (Hellweger, van Sebille and Fredrick 2014); however, this does not impede the genetic structuring of their global populations (Bottos et al. 2014;
Hellweger, van Sebille and Fredrick 2014). In microscopic green
algae, existence of ecologically determined lineages (or even
genotypes) was so far confirmed for only a handful of cases,
mostly in the temperate zone, e.g. Klebsormidium (Škaloud et al.
2014; Ryšánek, Hrčková and Škaloud 2015), Asterochloris (Škaloud
et al. 2015) and Trebouxia (Sadowska-Deś et al. 2014). In the polar
regions and also in tropical rainforests, green algae have been
poorly studied by highly variable molecular markers. No tropical endemics are known so far, but Dal Grande et al. (2014) detected genetically distinct lichen photobionts of the widespread
genus Dictyochloropsis in South America. In a pioneering study of
polar Klebsormidium, Ryšánek et al. (2016) agreed with the proposal of the moderate endemicity model (Foissner 2006), which
has also been confirmed for further protists (Boenigk et al. 2006;
Boo et al. 2010). Despite the wide agreement on the high microbial endemism in Antarctica (e.g. Vyverman et al. 2010), which is
often synonymous with mere phylogenetic uniqueness (e.g. De
Wever et al. 2009; Bahl et al. 2011; Strunecký, Elster and Komárek
2012; Jadoon et al. 2013; Škaloud et al. 2013), some authors warn
against overestimation due to the taxonomic ambiguity of microbial species (e.g. Fernandez-Carazo et al. 2011). Based on the
phylogenetic uniqueness, we might identify polar and tropical endemics, e.g. the Chlorella-like Arctic strain L24 (Shukla,
Kvı́derová and Elster 2011) or the Stichococcus-like Ecuadorian
clone KSK870SM6T. But we have noticed that some putative endemics could be redetected elsewhere, depending on the intensity of sampling (comp. the Antarctic Stichococcus-like strain
EO7-4). Similar observations have been reported for Antarctic
stramenopile algae (Rybalka et al. 2009, 2013) and cyanobacteria
(Taton et al. 2006; Namsaraev et al. 2010). Moreover, apart from
the natural vectors, airborne green algae might be introduced
to Antarctica by steadily increasing human activites (Convey
2010). One way or another, the intriguing genetic similarity
of some temperate and Antarctic Chlorellaceae suggests recurrent gene flow; understanding the speed of algal adaptation to harsh environments will change our view of microbial
biogeography.
11
SUPPLEMENTARY DATA
Supplementary data are available at FEMSEC online.
ACKNOWLEDGEMENTS
We thank the managers of the three Exploratories, Kirsten
Reichel-Jung, Swen Renner, Katrin Hartwich, Sonja Gockel, Kerstin Wiesner and Martin Gorke for their work in maintaining the plot and project infrastructure; Christiane Fischer and
Simone Pfeiffer for giving support through the central office,
Michael Owonibi for managing the central data base, and
Markus Fischer, Eduard Linsenmair, Dominik Hessenmöller, Jens
Nieschulze, Daniel Prati, Ingo Schöning, François Buscot, ErnstDetlef Schulze, Wolfgang W. Weisser and the late Elisabeth Kalko
for their role in setting up the Biodiversity Exploratories project.
Field work permits were issued by the responsible state environmental offices of Baden-Württemberg, Thüringen and Brandenburg (according to §72 BbgNatSchG). We thank Alena Lukešová
and Klára Řeháková for providing us with the cultures of polar Chlorella. We further thank Kathrin I. Mohr and Kristin C.
Pahlmann for their sequencing efforts and Maike Lorenz for depositing new algal strains in the Culture Collection of Algae at
the University of Göttingen (SAG).
FUNDING
The work was supported by a stipend of the German Academic
Exchange Service (DAAD) extended to L.H.; by the Priority Program 1374 ‘Infrastructure-Biodiversity-Exploratories’ of the German Research Foundation (DFG) [grant number DFG Fr905/16-1]
extended to T.F.; by the Project PAK 540 “Acceleration of Biodiversity assessment – development of tools and application in a
tropical mountain ecosystem” of the German Research Foundation (DFG) [grant number DFG Fr905/17-1] extended to T.F.; by
the German Federal Ministry of Education and Research [grant
number BMBF UKR 08/038] extended to T.F.; by Ministry of Education, Youth and Sports of the Czech Republic [grant number:
LM2010009, RVO67985939] extended to J.E. and by a grant of the
National Science Foundation (NSF) [grant number DEB-0529737]
extended to Louise A. Lewis, University of Connecticut, in collaboration with T.F.
Conflict of interest. None declared.
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