Taxonomic composition and diversity

We grouped the sequences into 451 OTUs, including 149, 46 and 5 OTUs assigned to Monothalamea, Globothalamea and Tubothalamea, respectively. Single-chambered, soft-walled monothalamous taxa are the most sequenced in each of our 42 deep-sea sediment samples, with 34 samples composed of more than 60% of monothalamid se-quences (Fig. 5.3A and Table 5.4: Supplementary information). The dominance of Monothalamea in our samples corroborates previous deep-sea foraminiferal research based on morphological (Gooday, 2002; Gooday et al., 2004b) and molecular methods (Lecroq et al., 2011). Multi-chambered, hard-shelled taxa belonging to Globothalamea are detected in all samples, but they are usually represented by few sequences (7.3%

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Figure 5.2: Read sequence data processing and quality filtering statistics. The number of sequences removed at each step of the paired-end reads assembly and quality filtering are shown for 384 different sets of parameters (boxplots). The numbers obtained using the set of parameters selected as a basis for further analyses are indicated (red points), as well as the sequence removal at each step of analysis (blue line). The initial (total) and final (filtered) numbers of reads are indicated as well as the number of reads removed because of ambiguous bases in the read forward (for-N) or in the reads reverse (rev-N), insufficient Phred quality for the read forward (for-Q) the reads reverse (rev-Q). Reads are removed if the contig is of insufficient quality on average (contig-Q), per base (contig-B) or too short (contig-L). Reads removed if the primer of the read forward is not found (no primerF) or found with too many errors (error primerF). The same holds for the primer of the reads reverse (no primer/error primerR). Reads removed if associated with an unexpected tag combination (mistags) or if too short (dimers).

on average), except in the fourth most sequenced sample with 32.8% of globothalamid sequences (S16). Approximately one quarter of the 31.9% of sequences that could be assigned to a Globothalamea family-rank clade correspond to Hormosinidae and Clade 3 (Fig. 5.3C and Table 5.5: Supplementary information). The later clade comprises op-portunistic species such as E. exigua that are globally distributed in deep-sea sediments

(Lecroq et al., 2009b). Conversely, taxa belonging to the Tubothalamea are rare and occur in only 15 samples (0.56% of sequences on average). In each station, the most abundantly sequenced taxa belong to two clades inferred mainly from eDNA sequences (Clade Y and ENFOR2) and with remarkably narrow variation in terms of number of reads per core ( Fig. 4A–C). It is noteworthy that the clade ENFOR2 is constituted of sequences collected in the Southern Ocean only, albeit in shallow and deep settings (Pawlowski et al., 2011b). Other environmental clades were formerly sequenced in the Southern Ocean deep-sea but comprise lineages also found in other geographic areas (Fig. 5.3B). Some OTUs were assigned to foraminiferal species well known to inhabit the deep Southern Ocean benthos, such as E. exigua, Oridorsalis umbonatus, Cibici-doides sp. or for the monothalamiids, Pelosina variabilis, Bathysiphon argenteus or Micrometula sp. Most of them were represented by rare sequences and their identifi-cation was facilitated by our extensive reference database comprising the sequences of specimens collected in the Southern Ocean (Brandt et al., 2007; Gooday et al., 2004a;

Cedhagen et al., 2009). Nevertheless, from 0.6% to 54.8% of the sequences could not be assigned to any higher-level taxon (18.4% on average), representing approximately half of the OTUs diversity (55.6%), as previously observed for Foraminifera in HTS conditions (Lecroq et al., 2011). These unassigned sequences correspond to the gen-uine foraminiferal DNA templates, as non-foraminiferal sequences are removed by our filtering approach. Indeed, the ca. 70 conserved bases between the forward primer and the 37f hypervariable region can be used to unambiguously authenticate foraminiferal sequences, and a BLASTn search (BLAST 2.2.25+ with default parameters) could only return matches against Foraminifera of the PR² database (Guillou et al., 2013). They may correspond to taxonomic groups such as Lagenida that are know to be common in the deep sea but not referenced in our database. Alternatively, they may correspond to the extracellular foraminiferal DNA preserved in the sediments (Lejzerowicz et al., 2013a) that accumulate damages on the primary structure of DNA (Gilbert et al., 2007) and thus could not be assigned to reference sequences.

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Unassigned Monothalamea Globothalamea Tubothalamea

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Proportion of reads (Monothalamea)

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Proportion of reads (other Foramininfera)

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C1 C2 C3 C4 C5 C6 C7 C8 C9 C10C11C12 C13C14

Figure 5.3: Taxonomic composition of nested samples before and after filtering based on OTU occurrences across core replicates: (A) absolute number of reads obtained for each sequenced, replicate sample. (B) Relative number of reads per taxon assigned to monotha-lamous Foraminifera. (C) Relative number of reads per taxon assigned to other, non-monothalamous Foraminifera. (D) Number of reads per OTU for each core sample before and after filtering based on OTU occurrences across replicates. The “total” number of reads found in at least on replicate are represented in red while the number of reads “found in all replicates” simultaneously are represented in blue. The numbers of OTUs in each case as indicated below the boxplots by colored number, respectively. (E) Relative number of reads per taxon assigned to monothalamous Foraminifera after filtering. (F) Relative num-ber of reads per taxon assigned to other, non-monothalamous Foraminifera. The samples are arranged by groups of three replicates for each sediment core. The three lines of colored bars on top annotate the samples origins, including the “Station” (upper bars; 3 colors), the MUC deployment or “Cast” (middle bars; 7 colors) and the sediment “Core” (bottom bars;

14 colors).

CladeG

Number of reads (log10)

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Number of reads (log10)

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Number of reads (log10)

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Number of reads (log10)

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Number of reads (log10)

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Figure 5.4: Abundance-ranked monothalamous Foraminifera for each station before and after filtering based on OTU occurrences across core replicates. For each taxon, the numbers of reads is represented in terms of total read numbers (italicized numbers) and distributed across cores (i.e. for each group of three replicates) as log10-transformed values (boxplots).

The numbers of reads are displayed separately for each station and for both the set of OTUs kept if present in at least one replicate (A: station 86, B: station 141, C: station 175) and the set of OTUs present in all core replicates (D: station 86, E: station 141, F: station 175).