Genus Richness of Microalgae and Cyanobacteria in Biological Soil Crusts from Svalbard and Livingston Island: Morphological Versus Molecular Approaches

Polar Biology https://doi.org/10.1007/s00300-018-2252-2

ORIGINAL PAPER

Genus richness of microalgae and Cyanobacteria in biological soil crusts from Svalbard and Livingston Island: morphological versus molecular approaches

Martin Rippin1 · Nadine Borchhardt2 · Laura Williams3 · Claudia Colesie4 · Patrick Jung3 · Burkhard Büdel3 · Ulf Karsten2 · Burkhard Becker1

Received: 20 September 2017 / Revised: 18 December 2017 / Accepted: 2 January 2018 © Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract Biological soil crusts (BSCs) are key components of polar ecosystems. These complex communities are important for terrestrial polar habitats as they include major primary producers that fx nitrogen, prevent soil erosion and can be regarded as indicators for climate change. To study the genus richness of microalgae and Cyanobacteria in BSCs, two diferent methodologies were employed and the outcomes were compared: morphological identifcation using light microscopy and the annotation of ribosomal sequences taken from metatranscriptomes. The analyzed samples were collected from Ny-Ålesund, Svalbard, Norway, and the Juan Carlos I Antarctic Base, Livingston Island, Antarctica. This study focused on the following taxonomic groups: Klebsormidiophyceae, Chlorophyceae, Trebouxiophyceae, Xanthophyceae and Cyanobacteria. In total, combining both approaches, 143 and 103 genera were identifed in the Arctic and Antarctic samples, respectively. Furthermore, both techniques concordantly determined 15 taxa in the Arctic and 7 taxa in the Antarctic BSC. In general, the molecular analysis indicated a higher microalgal and cyanobacterial genus richness (about 11 times higher) than the morphological approach. In terms of eukaryotic algae, the two sampling sites displayed comparable genus counts while the cyanobacterial genus richness was much higher in the BSC from Ny-Ålesund. For the frst time, the presence of the genera Chloroidium, Ankistrodesmus and Dunaliella in polar regions was determined by the metatranscriptomic analysis. Overall, these fndings illustrate that only the combination of morphological and molecular techniques, in contrast to one single approach, reveals higher genus richness for complex communities such as polar BSCs.

Keywords Biological soil crust · Eukaryotic algae · Cyanobacteria · Morphological identifcation · Metatranscriptomics

Introduction Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00300-018-2252-2) contains The Arctic region and Antarctica are extreme environments supplementary material, which is available to authorized users. defned by low temperatures year-round, low water availability, and large seasonal fuctuations with long periods of dark- * Martin Rippin martin.rippin@uni‑koeln.de ness during winter versus continuous irradiance in summer (Thomas et al. 2008a). Thus, plant cover is sparse and biodi- 1 University of Cologne, Botanical Institute, Zülpicher Str. versity is generally low, e.g. only two autochthonous fower- 47B, 50674 Cologne, Germany ing plants occur in Antarctica and these are mainly found in 2 Department of Applied Ecology & Phycology, University a few suitable areas along the coast (Thomas et al. 2008b; of Rostock, Albert‑Einstein‑Straße 3, 18059 Rostock, Pointing et al. 2015). These ecosystems are instead domi- Germany 3 nated by biological soil crusts (BSCs), which are the main Department of Plant Ecology & Systematics, University terrestrial primary producers and also important ecosystem of Kaiserslautern, PO‑BOX 3049, 67653 Kaiserslautern, Germany engineers (Evans and Johansen 1999; Yoshitake et al. 2010; Pointing and Belnap 2012; Williams et al. 2017). BSCs are 4 Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, 90183 Umeå, communities of eukaryotic algae, Cyanobacteria, lichens, Sweden bryophytes, as well as heterotrophic fungi and bacteria

Vol.:(0123456789)1 3 Polar Biology which colonize the surface and top millimeters of the soil cytochrome oxidase I (COX I), as well as internal simple (Belnap et al. 2001a; Belnap 2006; Büdel et al. 2016). Typi- sequence repeats (ISSR) of microsatellite regions (Buchheim cally occurring phyla of eukaryotic microalgae are Strep- and Chapman 1991; Wilmotte 1994; Doyle et al. 1997; An tophyta (Klebsormidiophyceae, Zygnematophyceae), Chlo- et al. 1999; Evans et al. 2007; Shen 2008; Sherwood et al. rophyta (Chlorophyceae, Trebouxiophyceae, Ulvophyceae) 2008; Hall et al. 2010). A huge pitfall is that the majority and Ochrophyta (Bacillariophyceae, Eustigmatophyceae, of microorganisms present in environmental samples, such Xanthophyceae) (Büdel et al. 2016). Certain crust organ- as BSCs, are unculturable (Ward et al. 1990; Schloss and isms, such as Cyanobacteria and eukaryotic algae, form a Handelsman 2005; Shi et al. 2009; Massana et al. 2014). To matrix which aggregates the living cells and soil particles overcome the limitation of cultivation, biodiversity assess- (Belnap 2006; Breen and Lévesque 2008). Hence, BSCs pro- ments can be performed by metabarcoding (Taberlet et al. vide protection against soil erosion and cryoturbation (Evans 2012; Yoon et al. 2016; Elferink et al. 2017). For this pur- and Johansen 1999). Moreover, BSCs enhance water and pose, total DNA is extracted from an environmental sample nutrient retention and the crust-associated Cyanobacteria fx (eDNA) and used as a template to generate an amplicon nitrogen which in turn creates improved conditions (fertiliza- mixture from a barcoding gene (Taberlet et al. 2012). Sub- tion) for seed germination and plant growth (Belnap et al. sequently, the generated PCR products are sequenced with a 2001b; Breen and Lévesque 2008). This accumulation of high-throughput sequencing (HTS) technique, such as Roche biomass forms the nutritional basis for higher trophic levels 454 or Illumina (Margulies et al. 2005; Bentley et al. 2008; such as the Svalbard reindeer (Rangifer tarandus platyrhyn- Taberlet et al. 2012). The identifcation of taxa is carried chus; Elster et al. 1999; Cooper and Wookey 2001). In gen- out by annotating the obtained sequence reads against an eral, BSCs are essential components of sparse ecosystems adequate database and sequence counts provide information such as the polar deserts. These communities are valuable about taxonomic abundance in the sample (Pawlowski et al. biological indicators for abiotic factors, ecological health 2011; Taberlet et al. 2012). However, metabarcoding also and climate change (Belnap et al. 2001b; Pushkareva et al. exhibits various pitfalls such as the introduction of sequence 2016). Due to global warming which particularly afects the errors during PCR, the design of suitable metabarcoding Arctic and the Antarctic Peninsula, an invasion by alien spe- primers, covering all taxa of interest, and, again, the need for cies is likely, which in turn might alter the BSC composition appropriate reference databases (Taberlet et al. 2012). For- (Frenot et al. 2005; Chown et al. 2012; Pushkareva et al. tunately, PCR-dependent bias and the dependency on single 2016; Lee et al. 2017). Hence, studying individual BSC barcodes can be avoided by exploiting shotgun metagenom- organisms, as well as the whole community, is important to ics and metatranscriptomics (Urich et al. 2008, 2014). Simi- fully address biodiversity, which is a prerequisite for moni- lar to metabarcoding, total nucleic acids are isolated from toring changes, as well as for predictions of future climate an environmental sample but the amplifcation step is omit- change (Pushkareva et al. 2016). ted (Urich et al. 2008, 2014). Instead, total DNA or cDNA The identification of autotrophic microorganisms in is directly applied to HTS and the resulting sequences are BSCs, such as eukaryotic algae and Cyanobacteria, is tra- assembled for any desired gene or transcript of interest, e.g. ditionally performed using light microscopy to examine the small ribosomal subunit RNA (SSU; Geisen et al. 2015). BSCs in situ, or through established cultures of selected This powerful approach enables a more reliable taxonomic crust organisms (Skinner 1932; Bischof and Bold 1963; identifcation compared to metabarcoding but relies on the Waterbury and Stanier 1978; Büdel et al. 2009). The taxa of availability of correctly determined sequence data (Urich interest are typically identifed using morphological traits et al. 2008; Klimke et al. 2011). Additionally, the isolation such as color, cell size and shape, or motility (Prescott 1964; of high-quality nucleic acids from soil samples, suitable Lind and Brook 1980; Cox 1996; John et al. 2002). The uti- for HTS, can be difcult due to the presence of humic sub- lization of light microscopy for identifcation is rather quick stances and exocellular RNase activity (Greaves and Wilson and inexpensive compared to molecular techniques (Misawa 1970; Rippin et al. 2016). 1999), but requires expert knowledge for many taxa as mor- The present study focused on a methodological compari- phological features are often difcult to recognize and distin- son of morphological and molecular approaches to analyze guish (Manoylov 2014). In addition, morphological features the microalgal and cyanobacterial genus richness of Arc- are not always stable as they can change in response to envi- tic and Antarctic BSCs. Studying polar BSCs improves the ronmental factors (Albrecht et al. 2017). As an alternative, knowledge on these microecosystems, their biodiversity a number of molecular markers, so-called barcodes, can be and ecological structure. In this study, the identifcation of used if unialgal cultures are available (Vieira et al. 2016). various groups of eukaryotic algae (Klebsormidiophyceae, Typical barcode sequences are the 16S/18S rRNA gene and Chlorophyceae, Trebouxiophyceae, Xanthophyceae) and the internal transcribed spacer (ITS) rDNA, the RuBisCO Cyanobacteria in one Arctic and one Antarctic BSC sam- large subunit (rbcL), the plastid elongation factor tufA, the ple was carried out. Eukaryotic and prokaryotic algae were

1 3 Polar Biology identifed using light microscopy in combination with suitable taxonomic keys. Furthermore, active species in the crust were targeted by applying a metatranscriptomic approach and algal as well as cyanobacterial taxa were identifed using fully assembled SSUs. The fnal results were combined and compared to assess the comprehensiveness of both techniques regarding the genus richness.

Materials and methods

Sampling

BSC samples were collected during expeditions to the Arctic and Antarctica in August 2014 and February 2015, respectively. The Arctic BSC (NÅ; cf. Rippin et al. 2016; Borchhardt et al. 2017; Williams et al. 2017, Jung et al. (in preparation)) was sampled in close vicinity to Ny-Ålesund, Svalbard, Norway (78°55′26.33″N 11°55′23.84″E; Fig. 1a). The research station Ny-Ålesund is located at Kongsforden and its climate is classifed as polar tundra (ET) according to the Köppen-Geiger system (Peel et al. 2007; Vogel et al. 2012). The temperature is low year-round with an annual average of − 4.5 °C, the highest monthly temperature is in July with 5.8 °C and the lowest in March with −12 °C (all temperatures are averaged from August 1993 to July 2011; Maturilli et al. 2013). The mean annual precipitation is 433 mm (Førland et al. 2011). Williams et al. (2017) found 90% of the area to be covered by BSCs. The Antarctic BSC (Gr1; cf. Williams et al. 2017) was collected close by the Spanish Juan Carlos I Antarctic Base at Livingston Island, South Shetland Islands (62°39′55.44″S 60°23′42.76″W; Fig. 1b). Similar to Svalbard, the South Shetland Islands can be regarded as polar tundra (ET) according to the Köp- pen-Geiger system (Pereira et al. 2006; Michel et al. 2014). The temperature at the Juan Carlos I Antarctic Base aver- ages − 1 °C annually, with the highest mean temperature (2.1 °C) in austral summer and the lowest mean temperature (− 4.6 °C) in winter (all temperatures are averaged from December 2001 to April 2003 and January 2007 to Febru- ary 2011; Bañón et al. 2013). The site receives on average 444.5 mm of precipitation annually (Bañón et al. 2013). The BSC coverage is approximately 43% with skeletal soils being predominant (Williams et al. 2017). BSC samples for metatranscriptomic analysis (1 g) were preserved using the Fig. 1 Overview map of sampling sites in the Arctic and Antarctica. LifeGuard™ Soil Preservation Solution (MO BIO Labora- a Svalbard in the Arctic. Samples were taken at Ny-Ålesund (NÅ). b Livingston Island which is part of the South Shetland Islands. Isolates tories, Carlsbad, CA, USA). were collected in close vicinity of the Juan Carlos I Antarctic Base (Gr1). Maps were created using snazzymaps.com Cultivation and microscopy

Enrichment cultures of eukaryotic algae were established Germany) containing Bold’s Basal Medium modified by transferring the sampled BSCs (NÅ, Gr1) onto solid according to Starr and Jefrey (1993). The cultures were 1.5% Difco™ Agar (Becton Dickonson, Heidelberg, kept at 15 °C, 30 μmol photons m−2 s−1 under a 16:8-h

1 3 Polar Biology light–dark regime. A detailed outline on the procedure Bioinformatics is given in Borchhardt et al. (2017b). The identifcation of genera or species was carried out by light microscopy The obtained raw reads (SRA bioproject PRJNA421095) (BX51, Olympus, Tokyo, Japan) with oil-immersion and were quality trimmed using Trimmomatics 0.35 (Bolger a 1000-fold magnifcation. Relevant identifcation keys are et al. 2014) and rRNA gene reads were fltered out using listed in Borchhardt et al. (2017b). A complete list of the SortMeRNA 2.1 (Kopylova et al. 2012) and the SILVA identifed Arctic algae (NÅ) was published by Borchhardt SSU NR Ref 119. All rRNA SSU reads were assembled et al. (2017a) and is used for comparison in the present using EMIRGE 0.61.0 (Miller et al. 2011) combined with study. the SILVA SSU NR Ref 128. The script emirge_amplicon. Cyanobacterial genera, present in the Arctic (NÅ) and py for single-end reads with the fag phred33 was run. Only Antarctic (Gr1) BSC were directly determined using light successfully assembled SSUs were considered. The assigned microscopy with oil immersion and a 630-fold magnifca- genera for Klebsormidiophyceae, Chlorophyceae, Trebouxi- tion using identifcation keys (Geitler 1932; Komárek and ophyceae, Xanthophyceae and Cyanobacteria were extracted Anagnostidis 1998, 2005). Cyanobacteria were additionally and compiled with an in-house R-script (Version 3.3.2). pre-cultivated on BG-11 medium at 15–17 °C, 20–50 µm photons m −2 s−1 under a 14:10-h light–dark cycle and identifed using suitable taxonomic keys as described by Williams Results et al. (2016). The data of Jung et al. (in preparation), which examined the cyanobacterial diversity of both sites, were The genus richness of phototrophic organisms in BSCs, iso- compared to the molecular dataset. lated from Svalbard (NÅ) and Livingston Island (Gr1), was assessed using morphological features and annotated environmental rRNA SSU sequences. We obtained 18,041,893, RNA isolation and sequencing 28,446,946 and 116,112,134 forward raw reads for sample NÅ and 62,235,763, 70,163,318 and 48,605,923 for Gr1. The Arctic sample (NÅ) was processed according to Rippin After quality trimming and rRNA SSU fltering we ended up et al. (2016) using the CTAB protocol, DNase I (Thermo with 8936, 7818 and 38,320 sequences for NÅ and 706,549, Fisher Scientifc, Waltham, MA, USA) and the RNeasy 1081,090 and 8646 for Gr1. This study focused on genera MinElute Cleanup Kit (Qiagen, Hilden, Germany). Due to belonging to the Klebsormidiophyceae, Chlorophyceae, Tre- low RNA yields, a total of six biological replicates were bouxiophyceae, Xanthophyceae or Cyanobacteria. All the extracted and combined to obtain three pooled replicates. taxa that were identifed are included in the supplementary RNA from three biological replicates, collected at Living- information (Online Resource 1). Additional taxonomic ston Island (Gr1), was isolated using the Spectrum™ Plant groups, for example Fungi, that were identifed in the molec- Total RNA Kit (Sigma-Aldrich, St. Louis, MO, USA), ular analysis were not further investigated in this study. treated with DNase I (Thermo Fisher Scientifc, Waltham, MA, USA) and purifed using the RNeasy MinElute Cleanup The Arctic BSC Kit (Qiagen, Hilden, Germany) as described by Rippin et al. (2016). Single samples yielded sufcient amounts of RNA. The Arctic sample contained 26 genera of eukaryotic algae All RNA samples were further processed by Eurofns and Cyanobacteria identifed by light microscopy while 132 Genomics (Ebersberg, Germany). The processing included were assigned by metatranscriptomic analysis. The average quality control utilizing the BioAnalyzer (Agilent Technolo- number of genera in the metatranscriptomic triplicates was gies, Santa Clara, CA, USA) and library preparation for both 71.7 ± 36.1 (SD). In total, 15 genera were determined by triplicates. Eukaryotic mRNA was enriched using oligo-(dT) both methodologies, compared to 117 and 11 genera which beads, fragmented and, subsequently, cDNA was synthesized were solely found by molecular and morphological identi- using random hexamers. Finally, Illumina compatible adapt- fcation, respectively (Fig. 2a). The utilized 18S reference ers were ligated. The libraries of the individual samples NÅ database contained sequences for 5 of the 11 genera iden- and Gr1 were applied to an Illumina HiSeq 2500, all tripli- tifed by morphology. Figures 3a and 4a display all gen- cates multiplexed on one lane, using 125 bp paired-end and era identifed with 1, 44, 25, 6 and 67 taxa belonging to single-end mode, respectively. For sample NÅ, the HiSeq Klebsormidiophyceae, Chlorophyceae, Trebouxiophyceae, Control Software 2.2.58, RTA 1.18.64 and bcl2fastq-1.8.4 Xanthophyceae and Cyanobacteria, respectively. The gen- were used while the detected signals from sample Gr1 were era Coccomyxa, Elliptochloris, Stichococcus (Trebouxi- processed by operating the HiSeq Control Software 2.2.38, ophyceae), Leptolyngbya, Microcoleus, Nostoc and Phor- RTA 1.18.61 and bcl2fastq-1.8.4 (Illumina, San Diego, CA, midium (Cyanobacteria) were found in the culture isolates USA). and in all three metatranscriptomes while Klebsormidium

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determined by morphology or sequence homology search, respectively (Fig. 2b). For three genera, identifed from culture isolates, rRNA reference sequences were present in the database. Regarding the diferent taxonomic groups, several genera were determined: one genus of Klebsormidiophy- ceae, 53 genera of Chlorophyceae, 28 genera of Trebouxi- ophyceae, six genera of Xanthophyceae and 16 genera of Cyanobacteria (Figs. 3b and 4b). The genera Coccomyxa (Trebouxiophyceae), Leptolyngbya, Nostoc and Phormid- ium (Cyanobacteria) could be determined morphologically and were identifed in all replicates used for the molecular approach. In addition, the genera Elliptochloris, Prasiola (Trebouxiophyceae), Heterococcus (Xanthophyceae) were determined by morphology and present in at least one out of three metatranscriptomic replicates. All three replicates prepared for molecular identifcation included the genera Ankistrodesmus, Bracteacoccus, Chlamydomonas, Chlo- romonas, Dunaliella, Hemifagellochloris, Monoraphidium, Oedogonium, Scenedesmus (Chlorophyceae), Botryococcus, Chlorella, Chloroidium, Heterochlorella, Nannochloris, Fig. 2 Comparing the molecular (blue, violet) and morphological Stichococcus (Trebouxiophyceae), Botrydiopsis (Xantho- (red, orange) approach in terms of identifed algal genera, both eukar- phyceae) and Chamaesiphon (Cyanobacteria); however, yotic and prokaryotic. The numbers divided by a slash represent the cultivation failed for these organisms. In contrast, cultures taxa present in the reference database (left) and all genera identifed Chlorococcum Heterotetra- (right). a NÅ; b Gr1 were established for the genera , cystis, Mychonastes, Neocystis* (Chlorophyceae), Desmo- coccus*, Muriella*, Myrmecia* (Trebouxiophyceae) and (Klebsormidiophyceae), Chlorococcum, Coelastrella, Pleurochloris* (Xanthophyceae) which are missing in the Desmodesmus, Lobochlamys (Chlorophyceae), Botrydiopsis, rRNA dataset. Five of these genera were not represented by Heterococcus (Xanthophyceae) and Stigonema (Cyanobac- 18S sequences in the used database (asterisk). teria) were identifed in at least one of the three replicates and by light microscopy. All three metatranscriptomes per Site comparison site included rRNA SSU sequences of the following genera, which could not be cultivated: Chloromonas, Dic- By comparing the Arctic and Antarctic crust samples tyococcus, Oedogonium, Scenedesmus (Chlorophyceae), we found 63 genera that were shared between both loca- Chlorella, Chloroidium, Dictyochloropsis (Trebouxiophy- tions (Fig. 5). However, the BSC collected from Svalbard, ceae), Anabaena, Aphanizonemon, Arthronema, Calothrix, included 80 microalgae and Cyanobacteria which were not Chroococcidiopsis, Crinalium, Cyanothece, Cylindrosper- shared with the Antarctic sample, although the sample from mum, Gloeobacter, Nodularia, Tolypothrix and Trichormus Livingston Island contained 40 additional taxa. The genus (Cyanobacteria). The morphology-based method identifed Klebsormidium occurred at both sites. The biggest portion the eukaryotic genera Gloeocystis*, Macrochloris*, Neocys- of chlorophycean (28) and trebouxiophycean (16) genera tis*, Tetracystis (Chlorophyceae), Desmococcus*, Koliella, was found in both samples. Regarding Xanthophyceae, both Lobosphaera (Trebouxiophyceae), Pleurochloris*, Pleuro- BSCs contained 6 genera of which 3 were shared between gaster*, Tribonema and Xanthonema (Xanthophyceae), of sites. The analyses of the Arctic and Antarctic samples which six were not represented by 18S rRNA gene sequences revealed 67 and 16 cyanobacterial genera to be present, in the reference database (asterisk). respectively. An overlap of 15 cyanobacterial genera was detected. The Antarctic BSC

A total of 15 genera, present in the Antarctic BSC, were Discussion determined based on morphology while molecular identifcation yielded 95 taxa. The genera mean value of the three Overall, the molecular survey of the BSC samples molecular replicates was 51.3 ± 5.7 (SD). Both techniques revealed a higher degree of genera richness for Chloro- found seven genera while 8 and 88 genera were solely phyceae, Trebouxiophyceae and Cyanobacteria compared

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Fig. 3 N Å All genera identifed − M a 2 3 belonging to either Klebsor- M Klebsormidium − midiophyceae ( ), Å Chlorophyceae, Trebouxiophy- N ceae or Xanthophyceae. The 1 N metatranscriptome (blue and M Å violet shades) is separated into − − Å C e three replicates while the cul- a e N ture isolates are represented by c Xanthonema C y idium Tribone h h m Klebsorm s a single category (red, orange). Pleurogastera l p Acutodesmu us o o Pleurochlo Ankistrodesm r ris o Chords connect the methodolo- h t Heterococcus Asteromonas rpha p n Atractomo h gies with the identifed genera. a Botr ydiopsis Barranca y

X Xylochloris c Genera displayed in gray were Bracteacoccus e

Tetrastrum a Bulbochaete oris e absent from the database used Symbiochl Carteria s Chaetopeltis for the metatranscriptomic Stichococcu Chaetopho ma Cha ra analyses. a NÅ; b Gr1 Raphidone raciochlor Characiopodiuis Prasiola Chl um amydo m Picochlor um Chlorococcummonas Pediastr ia adox Par Chloromona hlorella Coelast s Parac ocystis O ris Des rella m Dict odes y Nannochlo ania Dicty ochlor mu rv a s MicrothamnionMa a Dun ococcus is a Gloeoca Ha lie Lobosphaer lla Leptosiroliell Lobochlem K ys M a t Micro to is acroc coccu eochlorella Monoraphidium v Neoc am He Neocys spo hlor y s Oe s Elliptochloris Pa hlorosar ra is yxa P d va Pas r Po a ch ogo P ndo t Pyr is Quad Ro oly S yclad is lytom che n s n is

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to the culture and morphology-based methodology. Cyanobacteria, the Arctic crust sample exhibited a much However, the rRNA SSU identification did not recover higher variety compared to the sample collected at Liv- all genera determined by light microscopy. Regarding ingston Island.

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Fig. 4 All genera identifed 2 − M N belonging to Cyanobacteria. a Å Å N − The metatranscriptome (blue M 3 and violet shades) is separated into three replicates while the 1 culture isolates are represented M by a single category (red, − Å orange). Chords connect the N N

C methodologies with the genera Å Wilmottia is identifed. a NÅ; b Gr1 Westiellopsis ochlor Acary a konem Trichor sak mus Aero Trichodesmium Anabaena Tolypothrix Annamia Thermosynechococcus Aphanizomenon Synechococcus Aphanothece um Symplocastr Arthronema Symploca Arthrospi Brasilonemara Streptostemon Cald ora Stigonema ulina Caloth Spir rix owella Candid Sn a Chamaesiphonatus a Scytonem y Chlorogloeopsi ix othr Scytolyngb ia Chroococcidiopsis Schiz Rivular Chroococcus s Ch Chrysosproogloeocystis Rhabdogloea Coleofa

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Assessment of microalgal and cyanobacterial typically found in BSC communities and has been previ- genera richness ously found to colonize soils of both Livingston Island and Svalbard (Büdel 2001; Kastovská et al. 2005; Zidarova 2008; The genus Klebsormidium (Klebsormidiophyceae) is Borchhardt et al. 2017b). Our metatranscriptomic analyses

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Pushkareva et al. 2016; Borchhardt et al. 2017b). The genera Chlamydomonas, Coelastrella and Scenedesmus were previously identifed from Svalbard and Monoraphidium from Livingston Island (Matuła et al. 2007; Zidarova 2008; Pushkareva et al. 2016). Previous research describing the genera Ankistrodesmus and Dunaliella in polar habitats was not found; therefore, this is the frst study which confrms their presence in Svalbard and Livingston Island BSCs. Spe- cies of Chlorococcum were found in culture isolates prepared from both the Arctic and Antarctic BSCs as well as in the metatranscriptomic dataset generated from the NÅ sample. Kastovská et al. (2005) also found Chlorococcum in close vicinity to Ny-Ålesund and Borchhardt et al. (2017b) identifed this genus from samples collected from Ardley and King George Island, Antarctica. The genus Tetracys- tis was identifed from both BSC communities. Based on morphology, Tetracystis was found in the Arctic sample and metatranscriptomics confrmed its presence in the BSC from Livingston Island. Evidence exists that suggests Tetracys- tis colonizes the soil of Arctic and Antarctica ecosystems (Broady 1986; Giełwanowska and Olech 2012; Patova et al. 2015; Pushkareva et al. 2016). The following eukaryotic algae were only identifed in culture isolates: Gloeocystis and Macrochloris were present in isolates from the Arctic while Heterotetracystis and Mychonastes grew in the Ant- Fig. 5 Comparing the Arctic sample (NÅ) and the Antarctic BSC arctic enrichment cultures. The genus Neocystis was found in (Gr1) in terms of identifed total genera (gray) and genera belonging both samples. For almost fve genera their presence in polar to either Klebsormidiophyceae (dark green), Chlorophyceae (green), and alpine ecosystems has been reported previously (Matuła Trebouxiophyceae (light green), Xanthophyceae (yellow) or Cyano- bacteria (turquoise) et al. 2007; Zidarova 2008; Lukešová et al. 2010; Andreyeva and Kurbatova 2014; Patova et al. 2015; Pushkareva et al. 2016; Borchhardt et al. 2017b). confrmed the presence of this streptophytic taxon in the Both the Arctic and Antarctic metatranscriptomic data- polar regions, although Klebsormidium was not identifed sets and culture isolates contained the trebouxiophycean by light microscopy in the sample collected from Livingston genus Coccomyxa. Previous studies also found that this Island. genus is an inhabitant of Svalbard and Livingston Island Several chlorophycean genera were identifed in both the ecosystems (Matuła et al. 2007; Zidarova 2008; Pushkareva Arctic and the Antarctic samples by metatranscriptomic et al. 2016). Coccomyxa has a ubiquitous distribution and analysis: Ankistrodesmus, Chlamydomonas, Chloromonas, exhibits an extraordinary versatility, it occurs in terrestrial Coelastrella, Desmodesmus, Dictyococcus, Dunaliella, mats, in associations with soil, Fungi and bryophytes, and is Lobochlamys, Monoraphidium, Oedogonium and Scened- also planktonic in freshwater systems (Schmidle 1901; Vogel esmus. Coelastrella, Desmodesmus and Lobochlamys were 1955; Peveling and Galun 1976; Büdel 2001; Darienko also found in Arctic culture isolates. All the aforementioned et al. 2015). An ecophysiological study on three Cocco- genera are known to occupy terrestrial habitats from tem- myxa strains isolated from Antarctic BSC samples points to perate to cold climates (Büdel 2001; Borie and Ibraheem pronounced cold and drought tolerance (Pfaf et al. 2016). 2003; Lürling 2003; Tschaikner et al. 2007; Matuła et al. Rivasseau et al. (2016) even found a new species of Coc- 2007; Thorn and Lynch 2007; Uzunov et al. 2008; Zidarova comyxa, C. actinabiotis, in the cooling pool of a nuclear 2008; de Wever et al. 2009; Wu et al. 2016; Büdel et al. power plant. Thus, it is not surprising that members of this 2016; Pushkareva et al. 2016; Schulz et al. 2016; Borchhardt genus can colonize the extreme habitats which prevail in the et al. 2017b). For Chlamydomonas, Chloromonas, Coelas- polar regions. The green alga Elliptochloris generally occu- trella and Monoraphidium, earlier records exist confrming pies terrestrial habitats and associates with Fungi to form their occurrence in both polar regions; however, Dictyococ- lichens (Ettl and Gärtner 2014). Furthermore, this genus cus, Oedogonium and Scenedesmus have only been identi- was found in the Canadian Arctic and King George Island, fed from Arctic BSCs (Matuła et al. 2007; Zidarova 2008; Antarctica (Pushkareva et al. 2016; Borchhardt et al. 2017b).

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Both methodologies that were used in this study confrm that identifed by light microscopy only in the sample collected Elliptochloris is present in Svalbard and Livingston Island from Livingston Island. On the other hand, the genera Pleu- BSCs. In contrast, the genus Stichococcus was found in both rogaster, Tribonema and Xanthonema were only present in the Arctic and the Antarctic metatranscriptomic data and in isolates from the Arctic, while Pleurochloris was identifed isolates from Livingston Island but not from Svalbard. This in both samples by means of morphological methods. Pleu- alga has often been identifed as a member of BSC commu- rochloris, Tribonema and Xanthonema have been previously nities (Kastovská et al. 2005). Diferent species of Sticho- found in polar and alpine ecosystems (Matuła et al. 2007; coccus have been previously found to colonize the soils of Zidarova 2008; Patova et al. 2015; Pushkareva et al. 2016; both Livingston Island and Svalbard (Kastovská et al. 2005; Borchhardt et al. 2017b). However, the genus Pleurogaster Zidarova 2008). The green alga Prasiola was identifed had previously only been isolated from terrestrial habitats through both the metatranscriptomic and microscopic iden- outside polar regions (Stoyneva 2000). tifcation for Livingston Island terrestrial habitats. However, All metatranscriptomes and morphological analyses iden- only the Arctic metatranscriptomes showed evidence for tifed the cyanobacterial genera Leptolyngbya, Nostoc and Prasiola species. These fndings are supported by a previ- Phormidium from Svalbard and Livingston Island samples, ous survey on species richness which reported Prasiola from which was also reported by Pushkareva et al. (2016) and both Livingston Island and Svalbard (Matuła et al. 2007; Zidarova (2008), respectively. All genera occur in vari- Zidarova 2008). Based solely on the metatranscriptomic ous climates and in aquatic and terrestrial habitats (Rippka analysis, the genera Chlorella, Chloroidium and Nannochlo- et al. 1979; Büdel 2001; Casamatta et al. 2005; Dojani et al. ris were identifed in both the Arctic and Antarctic BSC 2014). The cyanobacterium Microcoleus also has a ubiqui- samples. Diferent species of Chlorella have been previously tous distribution, is often associated with BSCs and has been isolated from Svalbard and Livingston Island (Matuła et al. found in both Arctic and Antarctic environments (Büdel 2007; Zidarova 2008; Pushkareva et al. 2016). Vishnivets- 2001; Pushkareva et al. 2015). All metatranscriptomic data- kaya (2009) identifed Nannochloris in Siberian permafrost sets contained evidence of Microcoleus; however, it was only using molecular techniques, which suggests that this alga identifed from Svalbard through the morphological investi- can survive in extreme conditions. The genus Chloroidium gation. The genus Stigonema was identifed from Svalbard has been previously identifed in BSCs; however, it has not through both metatranscriptomic and morphological meth- been reported from polar habitats so far (Schulz et al. 2016; ods, which is in agreement with other reports (Matuła et al. Williams et al. 2016). Therefore, this study provides the frst 2007; Pushkareva et al. 2015). However, in contrast to our molecular record of Chloroidium in Arctic and Antarctic results Stigonema can also be found in Antarctic ecosys- ecosystems. The presence of Lobosphaera in the Arctic tems (Büdel 2001). The cyanobacterial genera Calothrix, and Antarctic BSC samples was supported by the morpho- Chamaesiphon, Chroococcidiopsis, Nodularia and Tolypo- logical and metatranscriptomic identifcation. Additionally, thrix, typically found in soil biocoenoses (Büdel 2001; Wang Giełwanowska and Olech (2012) identifed Lobosphaera as et al. 2015), were identifed in the Arctic and the Antarctic a photobiont of Antarctic lichens. Based on morphological sample using metatranscriptomics. Earlier studies, assessing identifcation, the genus Desmococcus was found in BSC the species richness of aeroterrestrial algae from Svalbard, samples from both sites, Koliella was identifed in isolates recorded all of these genera (Matuła et al. 2007; Pushkareva from the Arctic, while Muriella and Myrmecia grew in the et al. 2015, 2016). Antarctic enrichment cultures. These fndings are in accord- Interestingly, the Arctic crust community exhibited higher ance with previous studies reporting the presence of these cyanobacterial genus richness than the BSC from Livingston genera in polar and alpine habitats (Kawecka 1986; Takeuchi Island, while the genus richness of eukaryotic algae was 2001; Zidarova 2008; Lukešová et al. 2010; Andreyeva and similar. Our morphological and molecular analyses found Kurbatova 2014; Czerwik-Marcinkowska et al. 2015; Patova only 16 diferent genera of Cyanobacteria in the Antarctic et al. 2015; Pushkareva et al. 2016; Borchhardt et al. 2017b). sample compared to 67 in the Arctic. BSCs develop over The xanthophyte Heterococcus was identifed by both time and undergo changes in species composition (Büdel techniques from the Svalbard and Livingston Island sam- et al. 2009). Early successional stages of BSCs are usually ples. Pushkareva et al. (2016) and Borchhardt et al. (2017b) dominated by Cyanobacteria; however, bryophytes, lichens also identifed this genus in BSCs collected in the Cana- and/or liverworts may become the dominant groups in many dian Arctic and King George Island, Antarctica. The genus ecosystems (Büdel et al. 2009). Thus, diferences in cyano- Botrydiopsis is also typically found in BSCs and was pre- bacterial genus richness may be explained by the diferent viously isolated from other Arctic and Antarctic locations developmental stages of the communities. Furthermore, Wil- (Broady 1976; Büdel 2001; Pushkareva et al. 2016). Our liams et al. (2017) reported a lower abundance of cyanobac- metatranscriptomic analyses support the presence of this terial crusts on Livingston Island than at Ny-Ålesund, Sval- xanthophycean alga in the polar regions. However, it was bard. Our observation might also be linked to the reduced

1 3 Polar Biology occurrence of Cyanobacteria in these soil communities hardly anything is known about the underlying mechanisms. which was previously reported by Colesie et al. (2014a, b). BSCs are complex biocoenoses with many dependencies and co-dependencies which are not yet fully understood. Methodological diferences and difculties Another potential problem when comparing morphological and molecular methodologies is the false identifcation The identifcation of microalgae through metatranscriptom- of taxa due to ambiguous morphological traits (Wu et al. ics yielded a higher number of genera compared to the mor- 2001; Hodač et al. 2016). Some genera, such as Chlorella, phological determination of cultivated and directly observed are difcult to identify as their morphological characters are organisms. This is an indication for the lack of culturability limited and changeable depending on environmental condi- of most microorganisms associated with BSCs (Ward et al. tions (Bock et al. 2011; Hodač et al. 2016). This might be 1990; Schloss and Handelsman 2005; Shi et al. 2009; Mas- one possible explanation why the metatranscriptomic data sana et al. 2014). The successful cultivation of microalgae is confrmed the presence of Chlorella in the Arctic and the highly dependent on the applied growth conditions includ- Antarctic sample but the morphology-based identifcation ing temperature, irradiance and culture medium (Bold 1942; did not. However, it is more likely that the spatial heteroge- Hoham 1975; Singh and Singh 2015; Wu et al. 2016). For neity of the sample replicates collected for molecular and example, Hoham (1975) tested various cryophilic algae, light microscopic analysis caused some of the diferences isolated from alpine habitats in Washington, USA, and dis- (Concostrina-Zubiri et al. 2013; Kim et al. 2015). A clear covered that Chloromonas pichinchae reached the highest indication of heterogeneity is the difering results observed growth rate at 1 °C while Cylindrocystis brébissonii grew for the replicates of the same site in the molecular analysis. fastest at 10 °C. Therefore, the genus Chloromonas might For example, the genera Coelastrella, Chlorococcum and have been inhibited during our culturing procedure due to Chloromonas were found in one, two and three replicates of unsuitable temperatures. Regarding the growth medium, one the Arctic metatranscriptome, respectively. Furthermore, the single solution, that is suitable for all algae and Cyanobacte- high standard deviation of the averaged genera number for ria, does not exist (Bold 1942; Lee et al. 2014). Even though the molecular dataset of the Arctic BSC suggests sample het- the medium, used to establish the enrichment cultures, was erogeneity. Diferences in taxonomic determination may also optimized for a broad range of algae, certain strains may arise due to a lack of consensus in the algal taxonomy. Many not exhibit growth due to individual nutrient requirements. species concepts are still being revised based on molecular Some species of the genus Dunaliella, for example, prefer analyses rendering an unambiguous morphological identi- higher salt concentrations compared to other algae (Oren fcation difcult (Stoyanov et al. 2014; Champenois et al. 2005). The salt concentrations of Bold’s Basal Medium, 2015; Kawasaki et al. 2015; Škaloud et al. 2016). The genus used to establish the enrichment cultures, could have been Leptolyngbya, for example, was revealed to be heterogene- too low to enable the growth of Dunaliella. Similar reports ous and only recently split up into 15 new genera (Komárek exist for the cyanobacterium Chroococcidiopsis. Diferent 2016). strains of Chroococcidiopsis, isolated from diferent habi- Finally, we came across a number of genera which could tats, exhibited growth only if the medium was supplied with be identifed by light microscopy but not by the analysis the appropriate amount of sea salt (Cumbers and Rothschild of the rRNA SSU sequences. Regarding the genera Gloeo- 2014). However, growth parameters and culture medium cystis, Macrochloris, Neocystis, Desmococcus, Muriella, determine the culturability of microorganisms only to a Myrmecia, Pleurochloris and Pleurogaster, the correspond- certain extent. For many organisms, especially algae, the ing 18S sequences were missing from the database utilized co-cultivation of certain helper organisms (other algae, bac- in the course of identifcation. Incomplete and wrongly teria, etc.) is essential for growth and development (Jones annotated references are a major problem when perform- et al. 1973; Sanders et al. 2001; Vartoukian et al. 2010). ing molecular analyses of any kind and impair the correct Some Cyanobacteria, which form symbioses with eukaryotic determination of genera in environmental samples (Klimke algae, fail to grow in the absence of their partner (Thompson et al. 2011; Taberlet et al. 2012). The microbial dark matter, et al. 2012). It is likely that quorum sensing plays an impor- meaning the total of unculturable microorganisms, compli- tant role for these biotic interactions as certain signal mol- cates this matter even more (Solden et al. 2016). However, ecules from one organism infuence growth and development by studying metagenomics, metatranscriptomics and single of another (Poonguzhali et al. 2007; Williams 2007). Chi cell genomics researchers can overcome the limit of cultur- et al. (2017) studied the marine bacterium Ponticoccus sp. ability and supply the databases with novel sequences (Sol- PD-2 and how it controls the growth of the dinofagellates den et al. 2016). A number of algae, such as Chlorococcum, Alexandrium tamarense, Prorocentrum donghaiense and the Heterotetracystis, Mychonastes, Tetracystis, Koliella, Lobos- haptophyte Phaeocystis globosa. Although this clear indica- phaera, Tribonema and Xanthonema, were isolated from the tion of microalgae interacting with other microbes exists, BSC samples and were included in the reference database,

1 3 Polar Biology used for this study. However, they were not detected in the of higher quality. Thus, we recommend conjoined studies to metatranscriptomes. One possible explanation is the spatial validate fndings and maximize outcome. heterogeneity of the sample replicates (Concostrina-Zubiri et al. 2013; Kim et al. 2015). Furthermore, these organisms Acknowledgements This study was funded by the Deutsche Forschun- gsgemeinschaft (DFG) within the project ‘Polarcrust’ (BE1779/18-1, may have been inactive during sampling and were, there- KA899/23-1, BU666/17-1) which is part of the Priority Program 1158 fore, undetectable during total RNA analysis (Geisen et al. ‘Antarctic Research’. We also thank the AWIPEW station, the Instituto 2015). Many algae are known to develop resting cells to sur- Antártico Chileno, the Spanish Antarctic Committee and the Juan Car- vive unfavorable conditions such as drought (Holzinger and los I Antarctic Base for their logistic support. Sampling and research activities were approved by the German authorities (Umwelt Bunde- Karsten 2013; Li et al. 2015). Li et al. (2015) identifed 35 samt: Biological soil crust algae from the polar regions; 24.09.2014). diferent algae in resting stages, among them Chlorococcum which surrounds resting cells with a thin layer of mucus. A Compliance with ethical standards species of Mychonastes, M. desiccatus, may produce desiccation-resistant cysts if dried out (Margulis et al. 1988). Conflict of interest The authors declare that they have no confict of Massalski et al. (1994) isolated Lobosphaera from King interest. George Island, Antarctica, and studied its ultrastructure. The alga, colonizing volcanic rocks, was able to produce resting cysts with thickened cell walls to outlast detrimental conditions. Similar observations were made for Tribonema References bombycinum which produced akinetes in response to starva- tion (Nagao et al. 1999). 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