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.

Melting snow fields populated by aplanozygotes of the genus (Chlamydomonadales, Chlorophyta) are found in polar and alpine habitats. In the High Tatra Mountains (Slovakia), cells causing blooms of brownish-red snow designated as kol turned out to be genetically (18S, ITS1 and ITS2 rDNA, L) very closely related to (Chodat) Hoham et Mullet from the Austrian Alps. Therefore, is transferred into the latter taxon and reduced to a subspecies as subsp. Both exhibit a similar photosynthetic performance, thrive in similar habitats at open sites above timberline, but differ in astaxanthin accumulation and number of aplanozygote cell wall flanges. In a field sample of subsp. , polyunsaturated fatty acids formed nearly 50 % of total lipids, dominating in phospholipids and glycolipids. subsp. represents likely a variation of a common cryoflora species with distinct morphology.