Melting snow fields are an extremophilic habitat dominated by closely related Chlamydomonadaceae (Chlorophyta). Microscopy-based classification of these cryophilic microalgae is challenging and may not reveal the true diversity. High-throughput sequencing (HTS) allows for a more comprehensive evaluation of the community. However, HTS approaches have been rarely used in such ecosystems and the output of their application has not been evaluated. Furthermore, there is no consensus on the choice for a suitable DNA marker or data processing workflow. We found that the correct placement of taxonomic strings onto OTUs strongly depends on the quality of the reference databases. We improved the assignments of the HST data by generating additional reference sequences of the locally abundant taxa, guided by light microscopy. Furthermore, a manual inspection of all automated OTU assignments, oligotyping of the most abundant 18S OTUs, as well as ITS2 secondary structure analyses were necessary for accurate species assignments. Moreover, the sole use of one marker can cause misleading results, either because of insufficient variability within the locus (18S) or the scarcity of reference sequences (ITS2). Our evaluation reveals that HTS output needs to be thoroughly checked when the studied habitats or organisms are poorly represented in publicly available databases. We recommend an optimized workflow for an improved biodiversity evaluation of not only snow algal communities, but generally 'exotic' ecosystems where similar problems arise. A consistent sampling strategy, two- molecular marker approach, light microscopy-based guidance, generation of appropriate reference sequences and final manual verification of all taxonomic assignments are highly recommended.

Agroscope Schloss 1 8820 Wädenswil Switzerland GFZ German Research Centre for Geosciences Telegrafenberg 14473 Potsdam Germany

Department of Ecology Faculty of Science Charles University Prague Viničná 7 128 44 Prague 2 Czech Republic

GFZ German Research Centre for Geosciences Telegrafenberg 14473 Potsdam Germany School of Earth and Environment University of Leeds Woodhouse Lane Leeds LS2 9JT UK Department of Earth Sciences Free University of Berlin 12249 Berlin Germany

University of Applied Sciences Upper Austria Stelzhamerstraße 23 4600 Wels Austria

Zobrazit více v PubMed

Alanagreh L, Pegg C, Harikumar A, Buchheim M. Assessing intragenomic variation of the internal transcribed spacer two: Adapting the Illumina metagenomics protocol. PLoS One. 2017;12:e0181491. PubMed PMC

Anesio AM, Lutz S, Chrismas NAM, Benning LG. The microbiome of glaciers and ice sheets. npj Biofilms Microbiomes. 2017:3. PubMed PMC

Bengtsson-Palme J, Ryberg M, Hartmann M, Branco S, Wang Z, Godhe A, De Wit P, et al. Improved software detection and extraction of ITS1 and ITS2 from ribosomal ITS sequences of fungi and other eukaryotes for analysis of environmental sequencing data. Methods Ecol Evol. 2013;4:914–9.

Bock C, Medinger R, Jost S, Psenner R, Boenigk J. Seasonal variation of planktonic chrysophytes with special focus on Dinobryon . Fottea. 2014;14:179–90.

Bradley IM, Pinto AJ, Guest JS. Design and Evaluation of Illumina MiSeq- Compatible, 18S rRNA gene-specific primers for improved characterization of mixed phototrophic communities. Am Soc Microbiol. 2016;82:5878–91. PubMed PMC

Brandariz-Fontes C, Camacho-Sanchez M, Vilà C, Vega-Pla JL, Rico C, Leonard JA. Effect of the enzyme and PCR conditions on the quality of high-throughput DNA sequencing results. Sci Rep. 2015;5 8056. PubMed PMC

Broady PA. Six new species of terrestrial algae from Signy Island, South Orkney Islands, Antarctica. Br Phycol J. 1976;11:387–405.

Brown SP, Ungerer MC, Jumpponen A. A Community of Clones: Snow Algae Are Diverse Communities of Spatially Structured Clones. Int J Plant Sci. 2016:432–9.

Buchheim MA, Keller A, Koetschan C, F?rster F, Merget B, Wolf MM. Internal Transcribed Spacer 2 (nu ITS2 rRNA) Sequence-Structure Phylogenetics: Towards an Automated Reconstruction of the Green Algal Tree of Life. PLoS One. 2011;6:e16931. PubMed PMC

Caisová L, Marin B, Melkonian M. A Consensus Secondary Structure of ITS2 in the Chlorophyta Identified by Phylogenetic Reconstruction. Ann Anat. 2013;164:482–96. PubMed

Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer N, et al. QIIME allows analysis of high-throughput community sequencing data. Nat Methods. 2010;7:335–6. PubMed PMC

Chase MW, Fay MF. Ecology. Barcoding of plants and fungi. Science. 2009;325:682–3. PubMed

Cheung MK, Au CH, Chu KH, Kwan HS, Wong CK. Composition and genetic diversity of picoeukaryotes in subtropical coastal waters as revealed by 454 pyrosequencing. ISME J. 2010;4:1053–9. PubMed

Coleman A. Pan-eukaryote ITS2 homologies revealed by RNA secondary structure. Nucleic Acids Res. 2007;35:3322–9. PubMed PMC

Coleman AW. The significance of a coincidence between evolutionary landmarks found in mating affinity and a DNA sequence. Protist. 2000;151:1–9. PubMed

Coleman AW. ITS2 is a double-edged tool for eukaryote evolutionary comparisons. Trends Genet. 2003;19:370–5. PubMed

Coleman AW. Is there a molecular key to the level of “biological species” in eukaryotes? A DNA guide. Mol Phylogenet Evol. 2009;50:197–203. PubMed

Coleman AW, Suarez A, Goff LJ. Molecular delineation of species and syngens in Volvocacean green algae (Chlorophyta) J Phycol. 1993;30:80–90.

Darienko T, Gustavs L, Mudimu O, Menendez CR, Schumann R, Karsten U, Friedl T, et al. Chloroidium a common terrestrial coccoid green alga previously assigned to Chlorella (Trebouxiophyceae, Chlorophyta) Eur J Phycol. 2010;45:79–95.

Darty K, Denise A, Ponty Y. VARNA: Interactive drawing and editing of the RNA secondary structure. Bioinformatics. 2009;25:1974–5. PubMed PMC

Eddy SR. Hidden Markov models. Curr Opin Struct Biol. 1996;6:361–5. PubMed

Eren AM, Maignien L, Sul WJ, Murphy LG, Grim SL, Morrison HG, Sogin ML. Oligotyping: differentiating between closely related microbial taxa using 16S rRNA gene data. Methods Ecol Evol. 2013;4:1111–9. PubMed PMC

Frey B, Rime T, Phillips M, Stierli B, Hajdas I, Widmer F, Hartmann M. Microbial diversity in European alpine permafrost and active layers. FEMS Microbiol Ecol. 2016;92:1–17. PubMed

Grossmann L, Beisser D, Bock C, Chatzinotas A, Jensen M, Preisfeld A, Psenner R, et al. Trade-off between taxon diversity and functional diversity in European lake ecosystems. Mol Ecol. 2016;25:5876–88. PubMed

Hall JD, Fucíková K, Lo C, Lewis LA, Karol KG. An assessment of proposed DNA barcodes in freshwater green algae. Cryptogam Algol. 2010;31:529–55.

Hoham RW, Duval B. Microbial ecology of snow and freshwater ice with emphasis on snow algae. In: Jones HG, Pomeroy JW, Walker DA, Hoham R, editors. Snow Ecology. An Interdisciplinary Examination of Snow-Covered Ecosystems. Cambridge University Press; Cambridge: 2001. pp. 168–228.

Hoham RW, Roemer SC, Mullet JE. The life history and ecology of the snow alga Chloromonas brevispina comb. nov. (Chlorophyta, Volvocales)*. Phycologia. 1979;18:55–70.

Illumina n.d. 16S Metagenomic Sequencing Library Preparation Preparing 16S Ribosomal RNA Gene Amplicons for the Illumina MiSeq System. [last accessed March 20];2018 Available At: https://support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf.

Karsten U, Friedl T, Schumann R, Hoyer K, Lembcke S. Mycosporine-like amino acids and phylogenies in green algae: Prasiola and its relatives from the Trebouxiophyceae (Chlorophyta) J Phycol. 2005;41:557–66.

Kent WJ. BLAT—the BLAST-like alignment tool. Genome Res. 2002;12:656–64. PubMed PMC

Kol E. Kryobiologie; Biologie und Limnologie des Schnees und Eises. Schweizerbart’sche Verlagsbuchhandlung, Stuttgart. 1968

Komárek J, Nedbalová L. Green Cryosestic Algae. In: Seckbach J, editor. Algae and Cyanobacteria in Extreme Environments. Springer Netherlands; 2007. pp. 321–42.

Leliaert F, Verbruggen H, Vanormelingen P, Steen F, López-Bautista JM, Zuccarello GC, De Clerck O. DNA-based species delimitation in algae. Eur J Phycol. 2014;49:179–96.

Leya T. Snow algae: adaptation strategies to survive on snow and ice. In: Seckbach J, Oren A, Stan-Lotter H, editors. Polyextremophiles. Springer; 2013. pp. 401–23.

Lindner DL, Banik MT. Intragenomic variation in the ITS rDNA region obscures phylogenetic relationships and inflates estimates of operational taxonomic units in genus Laetiporus . Mycologia. 2011;103:731–40. PubMed

Lindner DL, Carlsen T, Henrik Nilsson R, Davey M, Schumacher T, Kauserud H. Employing 454 amplicon pyrosequencing to reveal intragenomic divergence in the internal transcribed spacer rDNA region in fungi. Ecol Evol. 2013;3:1751–64. PubMed PMC

Lutz S, Anesio AM, Edwards A, Benning LG. Microbial diversity on Icelandic glaciers and ice caps. Front Microbiol. 2015a;6:307. PubMed PMC

Lutz S, Anesio AM, Edwards A, Benning LG. Linking microbial diversity and functionality of arctic glacial surface habitats. Environ Microbiol. 2017;19:551–65. PubMed

Lutz S, Anesio AM, Field K, Benning LG. Integrated “Omics”, Targeted Metabolite and Single-cell Analyses of Arctic Snow Algae Functionality and Adaptability. Front Microbiol. 2015b;6:1323. PubMed PMC

Lutz S, Anesio AM, Raiswell R, Edwards A, Newton RJ, Gill F, Benning LG. The biogeography of red snow microbiomes and their role in melting arctic glaciers. Nat Commun. 2016;7 11968. PubMed PMC

Lutz S, McCutcheon J, McQuaid JB, Benning LG. The diversity of ice algal communities on the Greenland Ice Sheet as revealed by oligotyping. Microb Genomics. 2018:4. PubMed PMC

Matsuzaki R, Hara Y, Nozaki H. A taxonomic revision of Chloromonas reticulata (Volvocales, Chlorophyceae), the type species of the genus Chloromonas based on multigene phylogeny and comparative light and electron microscopy. Phycologia. 2012;51:74–85.

Matsuzaki R, Kawai-Toyooka H, Hara Y, Nozaki H. Revisiting the taxonomic significance of aplanozygote morphologies of two cosmopolitan snow species of the genus Chloromonas (Volvocales, Chlorophyceae) Phycologia. 2015;54:491–502.

McCormack JE, Hird SM, Zellmer AJ, Carstens BC, Brumfield RT. Applications of next-generation sequencing to phylogeography and phylogenetics. Mol Phylogenet Evol. 2013;66:526–38. PubMed

Mikhailyuk TI, Sluiman HJ, Massalski A, Mudimu O, Demchenko EM, Kondratyuk SY, Friedl T. New streptophyte green algae from terrestrial habitats and an assessment of the genus Interfilum (Klebsormidiophyceae, Streptophyta) J Phycol. 2008;44:1586–603. PubMed

Negrisolo E, Maistro S, Incarbone M, Moro I, Dalla Valle L, Broady PA, Andreoli C. Morphological convergence characterizes the evolution of Xanthophyceae (Heterokontophyta): evidence from nuclear SSU rDNA and plastidial rbcL genes. Mol Phylogenet Evol. 2004;33:156–70. PubMed

Novis PM. Ecology of the snow alga Chlainomonas kolii (Chlamydomonadales, Chlorophyta) in New Zealand. Phycologia. 2002;41:280–92.

Oyola SO, Otto TD, Gu Y, Maslen G, Manske M, Campino S, Turner DJ, et al. Optimizing illumina next-generation sequencing library preparation for extremely atbiased genomes. BMC Genomics. 2012;13:1. PubMed PMC

Procházková L, Remias D, Holzinger A, Rezanka T, Nedbalová L. Ecophysiological and morphological comparison of two populations of Chlainomonas sp. (Chlorophyta) causing red snow on ice-covered lakes in the High Tatras and Austrian Alps. Eur J Phycol. 2018a PubMed PMC

Procházková L, Remias D, Rezanka T, Nedbalová L. Chloromonas nivalis subsp. tatrae, subsp. nov. (Chlamydomonadales, Chlorophyta): re–examination of a snow alga from the High Tatra Mountains (Slovakia) Fottea. 2018b;18:1–18. PubMed PMC

Quast C, Pruesse E, Yilmaz P, Gerken J. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic acids. 2012 PubMed PMC

Remias D. Cell structure and physiology of alpine snow and ice algae. In: Lütz C, editor. Plants in Alpine Regions. Springer; Vienna: 2012.

Remias D, Holzinger A, Aigner S, Lütz C. Ecophysiology and ultrastructure of Ancylonema nordenskiöldii (Zygnematales, Streptophyta), causing brown ice on glaciers in Svalbard (high arctic) Polar Biol. 2012;35:1–10.

Remias D, Karsten U, Lütz C, Leya T. Physiological and morphological processes in the Alpine snow alga Chloromonas nivalis (Chlorophyceae) during cyst formation. Protoplasma. 2010;243:73–86. PubMed

Remias D, Lütz-Meindl U, Lütz C. Photosynthesis, pigments and ultrastructure of the alpine snow alga Chlamydomonas nivalis . Eur J Phycol. 2005;40:259–68.

Remias D, Pichrtová M, Pangratz M, Lütz C, Holzinger A. Ecophysiology, secondary pigments and ultrastructure of Chlainomonas sp. (Chlorophyta) from the European Alps compared with Chlamydomonas nivalis forming red snow. FEMS Microbiol Ecol. 2016:92. PubMed PMC

Remias D, Procházkova L, Holzinger A, Nedbalová L. Ecology, cytology and phylogeny of the snow alga Scotiella cryophila K-1 (Chlorophyceae) from the Austrian Alps. Accept with Revis Phycol. 2018 PubMed PMC

Remias D, Wastian H, Lütz C, Leya T. Insights into the biology and phylogeny of Chloromonas polyptera (Chlorophyta), an alga causing orange snow in Maritime Antarctica. Antarct Sci. 2013:1–9.

Schirmer M, Ijaz UZ, D’Amore R, Hall N, Sloan WT, Quince C. Insight into biases and sequencing errors for amplicon sequencing with the Illumina MiSeq platform. Nucleic Acids Res. 2015;43:e37. PubMed PMC

Schloss PD, Gevers D, Westcott SL. Reducing the effects of PCR amplification and sequencing Artifacts on 16S rRNA-based studies. PLoS One. 2011;6:e27310. PubMed PMC

Schmidt PA, Bálint M, Greshake B, Bandow C, Römbke J, Schmitt I. Illumina metabarcoding of a soil fungal community. Soil Biol Biochem. 2013;65:128–32.

Schultz J, Wolf M. ITS2 sequence-structure analysis in phylogenetics: A how-to manual for molecular systematics. Mol Phylogenet Evol. 2009;52:520–3. PubMed

Segawa T, Matsuzaki R, Takeuchi N, Akiyoshi A, Navarro F, Sugiyama S, Yonezawa T, et al. Bipolar dispersal of red-snow algae. Nat Commun. 2018;9 3094. PubMed PMC

Seibel PN, Müller T, Dandekar T, Schultz J, Wolf M. 4SALE--a tool for synchronous RNA sequence and secondary structure alignment and editing. BMC Bioinformatics. 2006;7:498. PubMed PMC

Seibel PN, Müller T, Dandekar T, Wolf M. Synchronous visual analysis and editing of RNA sequence and secondary structure alignments using 4SALE. BMC Res Notes. 2008;1:91. PubMed PMC

Simon U, Weiß M. Intragenomic variation of fungal ribosomal genes is higher than previously thought. Mol Biol Evol. 2008 PubMed

Stibal M, Elster J. Growth and morphology variation as a response to changing environmental factors in two Arctic species of Raphidonema (Trebouxiophyceae) from snow and soil. Polar Biol. 2005;28:558–67.

Terashima M, Umezawa K, Mori S, Kojima H, Fukui M. Microbial community analysis of colored snow from an alpine snowfield in Northern Japan reveals the prevalence of Betaproteobacteria with snow algae. Front Microbiol. 2017;8:1–13. PubMed PMC

Thornhill DJ, Lajeunesse TC, Santos SR. Measuring rDNA diversity in eukaryotic microbial systems: how intragenomic variation, pseudogenes, and PCR artifacts confound biodiversity estimates. Mol Ecol. 2007;16:5326–40. PubMed

Tragin M, Zingone A, Vaulot D. Comparison of coastal phytoplankton composition estimated from the V4 and V9 regions of 18S rRNA gene with a focus on photosynthetic groups and especially Chlorophyta. Environ Microbiol. 2017:1–41. PubMed

Vieira HH, Bagatini IL, Guinart CM, Vieira AAH. tufA gene as molecular marker for freshwater Chlorophyceae. ALGAE. 2016;31:155–65.

White TJ, Bruns T, Lee S, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, editors. PCR Protocols. Elsevier; 1990. pp. 315–22.

Wolf M, Chen S, Song J, Ankenbrand M, Müller T. Compensatory Base Changes in ITS2 Secondary Structures Correlate with the Biological Species Concept Despite Intragenomic Variability in ITS2 Sequences - A Proof of Concept. PLoS One. 2013;8:1–5. PubMed PMC

Xiao X, Sogge H, Lagesen K, Tooming-Klunderud A, Jakobsen KS, Rohrlack T. Use of High Throughput Sequencing and Light Microscopy Show Contrasting Results in a Study of Phytoplankton Occurrence in a Freshwater Environment. PLoS One. 2014;9:e106510. PubMed PMC

Yao H, Song J, Liu C, Luo K, Han J, Li Y, Pang X, et al. Use of ITS2 Region as the Universal DNA Barcode for Plants and Animals. PLoS One. 2010;5:e13102. PubMed PMC

Zou S, Fei C, Wang C, Gao Z, Bao Y, He M, Wang C. How DNA barcoding can be more effective in microalgae identification: a case of cryptic diversity revelation in Scenedesmus (Chlorophyceae) 2016 PubMed PMC

Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 2003;31:3406–15. PubMed PMC

Novel insights in cryptic diversity of snow and glacier ice algae communities combining 18S rRNA gene and ITS2 amplicon sequencing

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

The snow alga Chloromonas kaweckae sp. nov. (Volvocales, Chlorophyta) causes green surface blooms in the high tatras (Slovakia) and tolerates high irradiance

Ecophysiological and ultrastructural characterisation of the circumpolar orange snow alga Sanguina aurantia compared to the cosmopolitan red snow alga Sanguina nivaloides (Chlorophyta)

Cysts of the Snow Alga Chloromonas krienitzii (Chlorophyceae) Show Increased Tolerance to Ultraviolet Radiation and Elevated Visible Light