Replicate heterogeneity and common sequences

Large variations exist among the sample replicates of a same sediment core (Fig. 5.3B and 5.3C). On average, only 24.8% of all the OTUs found in a sediment core are occur-ring in all three subsample replicates simultaneously (from 12.25% to 30% depending on the core). In 10 out of 14 cores, there are more OTUs found in all replicates than OTUs exclusive to single replicates (Fig. 5A). The OTUs not shared by all replicates are mostly limited to one replicate rather than shared by pairs of replicates (Tables 5.6 and 5.7: Supplementary information). This is exemplified in core C4 by OTUs belonging to Clade D, Clade Y and the environmental clade ENFOR1 being abun-dantly sequenced but not in all three replicate (Table 5.5 Supplementary information).

The fact that this particular core is the least sequenced (Fig. 5.3A) suggests that the

presence of OTUs across replicates is due to sequence under sampling. However, this is clearly not the case as it is possible to find abundant taxa abundantly sequenced in only two sample replicates of well-sequenced cores (e.g. C1, C2, C5 or C9) (Table 5.7: Supplementary information). It is well known that the diversity is overestimated because of the PCR amplification step of eDNA sequencing studies (Huse et al., 2010;

Schloss et al., 2011; Lee et al., 2012). Here, the diversity is inflated at the level of the subsample replicate since it is the sampling unit processed for DNA extraction, PCR enrichment and sequencing. It has been questioned whether the diversity produced by several PCR should be combined (Schmidt et al., 2013) or whether the OTUs found at the intersection of PCR replicates represents dominant, genuine diversity (Zhou et al., 2011).

The number of OTUs representing the diversity found in the union of all subsam-ple replicates of a sediment core strongly decreases if only the shared OTUs that are found in all replicates simultaneously are kept. This sample replicate-intersection filter reduces the total OTUs richness of a core ranging from 90 to 179 OTUs to only 12 to 48 shared OTUs (Fig. 5.3D). For each core, the OTU diversity present in all repli-cates is dominated by the taxa that were highly sequenced in at least one replicate, as evidenced by ENFOR9 in the core C6 (Fig. 5.3F) and by Hormosinidae in the core C12 (Fig. 5.3F). This observation corroborates the results of (Zhou et al., 2011), who performed PCR replicates on the same sample, suggesting that an OTU abundantly sequenced in a gram of sediment also has good chances to be sequenced in another gram from the same core and certainly represent truly dominant species. Moreover, the dominance of the abundant taxa remains unchanged after the removal of sequences not found simultaneously in all replicates of each core, confirming the ability to de-tect the presence of important species at the scale of the sediment core sub-sample (Fig. 5.4D–F). This means that the most abundant species do not seem to be affected by the PCR amplification biases that skew the relative proportions of sequences (Dea-gle et al., 2013).

The majority of the sequences are assisgned to OTUs cosmopolitan to the three stations, both before (90.6%) and after (76.5%) filtering, while only up to 0.6% and up to 4.4% of reads are exclusively found in one station respectively (Fig. 5.5B and 5.5C).

Half of the 157 OTUs found in all stations are unassigned, while after filtering this

Station 86

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Globothalamea Monothalamea Tubothalamea Unassigned

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Figure 5.5: Distribution of OTUs and reads among and within sampling scales before and after the filtering based on OTU presence in all replicates simultaneously: (A) for each station is shown the number of OTUs found across sample replicates of each core (inter-replicate), found across cores of each multicorer cast (inter-core) found across multicorer casts of each station (inter-cast). The cores are labeled from C1 to C14 as in Fig. 3. The stars indicate comparisons of filtered OTUs. (B) Inter-station distribution of the OTUs formed using the total dataset. (C) Inter-station distribution of the OTUs resulting from the filtering based on OTU presence in all replicates simultaneously. The number of sequenced reads (italicized numbers) and OTUs (numbers within circle charts) are indicated, as well as the higher-level Foraminifera diversity, both in terms of reads (outer circle charts) and OTUs (inner circle charts).

proportion is reduced to 7 out of 33 OTUs and the OTUs exclusively found in one sta-tion accumulate more reads. After applying the filter based on the presence of OTUs across all sediment replicates, broad beta-diversity analyses will tend to better sepa-rate samples as less OTUs contribute to community similarities. Moreover, it seems that controlling the distribution of reads at the core level by such filtering can help mitigating potential contamination events to which HTS is extremely sensitive (Orsi et al., 2013), including potential cross-contaminations originating from the mistagging phenomenon (Carlsen et al., 2012; Carew et al., 2013). For example, the OTU as-signed to the shallow water genus Elphidium was discarded because it was present in only one replicate. On the contrary, the OTUs assigned to the rare Hauerinidae family, abundantly sequenced only in one station but detected in the two other stations with very few sequences were retained, in agreement with morphological observations of the presence of this family in the deep-sea samples.

Inter-replicate variability mainly occurs in terms of relative sequence abundances, with numerous OTUs represented by few sequences (Fig. 5.3B and 5.3C). Indeed, the 320 OTUs not found in all replicates are represented by 27.1 reads per core, on average, while it reaches 1083.3 for the 131 OTUs found in all replicates of a core. If all the OTUs assigned to the same family-rank taxon are merged, this number remains as low as 322 reads per taxon on average for 29 taxa, and never exceeds 150 for 20 of these taxa (Table 5.7; Supplementary information). The fact that some OTUs dominate in a replicate but are only detected by few sequences in another replicate of the same core may be due to species genomes characteristics. Indeed, it is assumed that the large variations in the number of ribosomal DNA copies influence diversity estimates (Zhu et al., 2005; Not et al., 2009), as discussed for Foraminifera (?). Moreover, the copies corresponding to pseudogenes (Glöckner et al., 2014) or extracellular DNA (Corinaldesi et al., 2011) can readily be PCR amplified and sequenced, artificially increasing the observed diversity.

As shown in the study of deep-sea nematodes (Danovaro et al., 2013b), the mi-crohabitat heterogeneity greatly influences the diversity found in the replicates. The same is observed in our samples. Substantial diversity is missed in every individual replicate and even at the MUC cast and station levels, with comparable fractions of rare OTUs present in filtered or non-filtered samples (Fig. 5.5). Among the OTUs that are too rare to occur in all replicates we find some common deep-sea genera, such as

Epistominella, Cibicidoides Pelosina, Bathysiphon, orMicrometula. Their presence in few replicates only may reflect the patchiness of the distribution of their rare represen-tatives. However, we cannot exclude that some of these sequences originated from the extracellular DNA known to be extremely abundant in deep-sea sediments (Dell’Anno and Danovaro, 2005). It is only by targeting RNA instead of DNA that their living state could be confirmed (Lejzerowicz et al., 2013b). Using RNA-based sequencing, surprisingly similar microbial communities were found active in the sediments of the investigated stations, exposed to contrasting levels of particulate organic matter (Ruff et al., 2014), suggesting comparable particulate organic matter qualities. Small-scale patterns of meiofauna density are controlled by the primary production, but the link between organic matter fluxes and the establishment and sustainment of deep-sea di-versity remains largely unknown (Lins et al., 2014).