Phylogenetic differences in OTU diversity
Since several studies have shown that different ecotypes exist especially in many phytoplankton groups in the Arctic (i.e., Lovejoy et al., 2007; Monier et al., 2013; Onda, Medrinal, Babin, Thaler, & Lovejoy, 2017; Joli et al., 2018), we thoroughly evaluated the OTU diversity amplified from the sediment trap samples by the three different primer pairs. Although all primer pairs were able to amplify sequences belonging to Chlorophyta, Bacillariophyta and Haptophyta, significant differences in OTU diversity were observed at the phylogenetic level using the EPA-RAxML approach.
Among the four classes of Chlorophyta, only Mamiellophyceae was amplified from all samples and dominated the groups across datasets. However, the Wolf964 sequences significantly differed in the phylogenetic placement of its HTS fragments compared to Stoeck and Wolf938 primer sets (Figure 4). Consistent with the comparison at higher taxonomic level, the Wolf964 dataset contained lower numbers of Mamiellophyceae related-OTUs (6 OTUs) compared to Stoeck (33 OTUs) and Wolf938 (47 OTUs) datasets (Figure 4A). Wolf964 also did not amplify the summer ecotype Clade C closest to CCMP1195, which on the other hand was abundant in Stoeck and Wolf938. Placements of some potential Clades B and C sequences in the Wolf964 library were also not well supported in the tree (Figure 4A). All of the three primer pairs however were able to amplify sequences from Clade Ea, most similar to the reference sequence CCMP2099 (Micromonas polaris ).
For the Haptophyceae, although binned taxonomic abundance did not differ significantly across datasets (Figure 3), the number of OTUs identified and the classification at the lower taxonomic- and sequence-levels however differed significantly. Consistent with the in silicotests, Stoeck primers were only able to amplify 2 sub-phyla, including Isochrysidales (Emiliania ) and Phaeocystales (Phaeocystis ) totaling to only 8 OTUs, while Wolf964 also amplified sequences belonging to Prymnesiales (mainly Chrysochomulina ) in addition to the first 2 described genera, also with a total of 8 OTUs. In contrast, the Wolf938 amplified the highest number of different OTUs with 2 from Isochrydales (Isochrysis and Emiliania ), 3 OTUs from Prymnesiales (Chrysochomulina ), and with 18 OTUs from Phaeocystales (Phaeocystis ). Further, EPA approach revealed that the Phaeocystales- related sequences even clustered into different clades within the genus, mostly belonging to P. pouchetii (Figure 4B).
Another significant difference in the observed amplification biases was on Bacillariophyta. All tested primers were able to amplify sequences from major diatom groups known to occur in the Arctic including the class Coscinodiscophyceae, and mostly from Mediophyceae but with significant variability. Surprisingly, Wolf964 and Wolf938 only amplified a total of 15 and 12 OTUs, respectively while that of Stoeck, which performed poorly in the mock community test detected 26 OTUs. The dominant genus Chaetoceros belonged to two phylotypes, namelyC. brevis/debelis and C. gelidus/socialis complexes (Figure 4C).