Carbon isotope excursions during the late Miocene recorded by lipids of marine Thaumarchaeota, Piedmont Basin, Mediterranean Sea

Group I mesophilic Thaumarchaeota fix dissolved inorganic carbon (DIC), accompanied by a biosynthetic fractionation factor of ∼ 20‰. Accordingly, the δ 13 C signature of their diagnostic biomarker crenarchaeol was suggested as a potential δ 13 C DIC proxy in marine basins if input from nonmarine Thaumarchaeota is negligible. Semi-enclosed basins are sensitive to carbon-cycle perturbations, because they tend to develop thermohaline stratification. Water column stratification typified the semi-enclosed basins of the Mediterranean Sea during the late Miocene (Messinian) salinity crisis (5.97–5.33 Ma). To assess how the advent of the crisis affected the carbon cycle, we studied sediments of the Piedmont Basin (northwestern Italy), the northernmost Mediterranean subbasin. A potential bias of our δ 13 C DIC reconstructions from the input of soil Thaumarchaeota is discarded, since high and increasing branched and isoprenoid tetraether (BIT) index values do not correspond to low and decreasing δ 13 C values for thaumarchaeal lipids, which would be expected in case of high input from soil Thaumarchaeota. Before the onset of the crisis, the permanently stratified distal part of the basin hosted a water mass below the chemocline with a δ 13 C DIC value of approximately − 3.5‰, while the well-mixed proximal part had a δ 13 C DIC value of approximately − 0.8‰. The advent of the crisis was marked by 13 C enrichment of the DIC pool, with positive δ 13 C DIC excursions up to + 5‰ in the upper water column. Export of 12 C to the seafloor after phytoplankton blooms and limited replenishment of remineralized carbon due to the stabilization of thermohaline stratification primarily caused such 13 C enrichment of the DIC pool


INTRODUCTION
Marine group I (MGI) mesophilic Thaumarchaeota are among the dominant archaea in marine environments, where their occurrence can be traced by crenarchaeol, the thaumarchaeal-specific isoprenoid glycerol dialkyl glycerol tetraether (iGDGT; e.g., Besseling et al., 2020).In modern marine sediments, the δ 13 C values of the cyclohexane ring-containing tricyclic biphytane (Bp-cren) derived from ether cleavage of crenarchaeol typically fall between −23‰ and −18‰ (δ 13 C Bp-cren ; Schouten et al., 2013).These values originate from autotrophic fixation of dissolved inorganic carbon (DIC) as bicarbonate, with a biosynthetic fractionation factor (ε) of ∼20‰ ( Könneke et al., 2012).Accordingly, δ 13 C Bp-cren was suggested as a potential (paleo-)δ 13 C DIC proxy, assuming that ancient MGI Thaumarchaeota had the same metabolism as their modern heirs (Schouten et al., 2013).However, the uptake of organic carbon or the input of crenarchaeol from marine Euryarchaeota was claimed to compromise δ 13 C DIC estimates (Pearson et al., 2016).Such concerns were recently discarded (cf.Pearson et al., 2019;Besseling et al., 2020), although it has been put forward that ε values could depend on growth rate and carbon dioxide concentration, resulting in deviations of up to ±2‰ between the measured and the reconstructed δ 13 C DIC values of modern seawater (cf.Hurley et al., 2019).Another potential bias for δ 13 C DIC reconstructions is riverine input of crenarchaeol sourced by soil-and river-dwelling Thaumarchaeota to marine basins (Pearson et al., 2016).Elling et al. (2019) disputed the effect of such bias, even under high input of soil-derived crenarchaeol to ancient marine sediments.Therefore, δ 13 C Bp-cren values could, indeed, be a powerful (paleo-)δ 13 C DIC proxy, as demonstrated by successful applications in tracing past carbon-cycle perturbations (Kuypers et al., 2001;Schoon et al., 2013;Elling et al., 2019).
Carbon-cycle perturbations are common for modern semi-enclosed basins, which tend to develop thermohaline stratification, altering the δ 13 C DIC of the water column (e.g., Fry et al., 1991).Water masses below the chemocline tend to become 13 C depleted due to pronounced organic matter recycling, resulting in δ 13 C DIC values as low as −19‰ (e.g., van Breugel et al., 2005).Conversely, seasonal eutrophication and preferential 12 CO 2 loss via enhanced degassing in surface waters can produce 13 C enrichment, with δ 13 C DIC values as high as +16.5‰ in lacustrine environments (e.g., Stiller et al., 1985;Oren et al., 1995).Akin to modern basins, thermohaline stratification may have caused the alteration of δ 13 C DIC pools also in ancient semienclosed basins (e.g., Schoon et al., 2013).
In the late Miocene, the Mediterranean Basin developed thermohaline stratification (García-Veigas et al., 2018) due to its close-to-complete tectonic isolation from the global ocean during the Messinian salinity crisis (MSC; 5.97-5.33Ma).Although the global carbon cycle was likely affected by this event (e.g., Capella et al., 2019), the effect of the advent of the MSC on the δ 13 C DIC pool of the late Miocene Mediterranean water column remains unassessed.To fill this gap, we conducted a case study on the Piedmont Basin (northwestern Italy), the northernmost subbasin of the late Miocene Mediterranean Sea (Dela Pierre et al., 2011).There, MGI Thaumarchaeota represented the main group of planktonic archaea at the onset of the MSC (Natalicchio et al., 2017;Sabino et al., 2021).
After assessing potential biases in the applicability of δ 13 C Bp-cren values as paleo-δ 13 C DIC proxies, we reconstructed the δ 13 C DIC of the stratified water body in proximal (Pollenzo section) and distal (Govone section) sectors of the margin of the Piedmont Basin.The approach used in this study shows great promise to decipher ancient carbon-cycle perturbations.

MATERIALS AND METHODS
We analyzed five lithologic cycles (shalemarl couplets) deposited across the MSC onset (5.97 Ma) at a water depth >200 m in a more proximal (Pollenzo; 43 samples) and a distal (Govone; 14 samples) position along the southern margin of the Piedmont Basin (Figs. 1 and 2; for details, see the Supplemental Material 1 ).The cycles reflect astronomically driven moister (shales) and drier (marls) climate oscillations (cf.Natalicchio et al., 2019;Sabino et al., 2020).In the studied sections, the MSC onset was placed at the base of marls of cycles Pm5 (Pollenzo; Natalicchio et al., 2019) and Gm30 (Govone;Gennari et al., 2020;Fig. 2).An aliquot of the total lipid extract of the 57 samples was used to obtain data on iGDGTs and branched GDGTs (bGDGTs), analyzed through high-performance liquid chromatography-mass spectrometry.We calculated the branched and isoprenoid tetraether (BIT) index and the iGDGT-2/iGDGT-3 ratio ([2]/[3] ratio) to assess input from soil Thaumarchaea and the relative contributions of upper-water-column versus deeper-watercolumn thaumarchaeal communities, respectively (Hopmans et al., 2004;Kim et al., 2015).Ether-bound isoprenoids were released from iGDGTs by treating another aliquot of the extracts with HI/LiAlH 4 , while the asphaltene fraction of 21 samples (Pollenzo: 7 samples; Govone: 14 samples) was desulfurized to release sulfur-bound compounds from macromolecules.The hydrocarbon fractions obtained from ether cleavage and desulfurization were analyzed using gas chromatography-mass spectrometry.The δ 13 C analyses of ether-cleaved biphytanes (Bp) and desulfurized phytane and C 27 to C 29 steranes obtained after column chromatography were performed on 41 samples (Pollenzo: 27 samples; Govone: 14 samples).The δ 13 C values are reported in per mil (‰) versus the Vienna Peedee belemnite (V-PDB) standard, and the average analytical standard deviation was 0.4‰ (see the Supplemental Material for details).

RESULTS AND DISCUSSION δ 13 C Bp-cren Values as a Robust δ 13 C DIC Proxy
The BIT index suggests high (0.4-1.0, proximal sector, Pollenzo) to moderate (0.3-0.7, distal sector, Govone) soil-derived organic matter input into the Piedmont Basin at the advent of the MSC (Fig. 2A).A high BIT may imply high input of crenarchaeol sourced from group I soil Thaumarchaeota to marine sediments (Weijers et al., 2006), hampering the applicability of crenarchaeol and its derivative Bp-cren as a δ 13 C DIC proxy in this case.The δ 13 C Bp-cren values from MGI Thaumarchaeota range from −23‰ to −18‰ in modern marine sediments (Schouten et al., 2013, and references therein).In contrast, the δ 13 C Bp-cren values of soil Thaumarchaeota are approximately −30‰ in soils dominated by C 3 plants and −23‰ in soils where C 4 plants prevail (Weijers et al., 2010).Because C 3 plants dominated the Piedmont Basin hinterland in the late Messinian (Bertini and Martinetto, 2011), a bias from soil archaea should result in high and increasing BIT values corresponding to low and decreasing δ 13 C Bp-cren values.However, the reverse trend was found for the studied samples, with lower δ 13 C Bp-cren values occurring when BIT values decreased, and vice versa (Figs.2B  and 3).The input of soil thaumarchaeal lipids was therefore apparently negligible.The bGDGTs were likely rather sourced from bacteria dwelling in rivers, coastal marine sedi-ments, and in oxygen-deficient waters (e.g., Liu et al., 2014;Crampton-Flood et al., 2021).This agrees with increasing BIT values coinciding with higher river discharge and an expansion of oxygen-deficient waters toward the margin of the Piedmont Basin after the MSC onset (Natalicchio et al., 2019;Sabino et al., 2020Sabino et al., , 2021)).Because Thaumarchaeota have also been reported to dwell in rivers (e.g., Kim et al., 2007;Yang et al., 2013), river-derived crenarchaeol with increasing fluvial discharge can also affect δ 13 C DIC reconstructions of seawater.Today's rivers draining the former catchment area of the Piedmont Basin are typified by a mean δ 13 C DIC value of −8‰ (Marchina et al., 2016).Assuming a similar value for the late Miocene and an ε = 20‰ (Könneke et al., 2012) for fluvial Thaumarchaeota, a trend toward 13 C depletion of Bp-cren with increasing river discharge is likely.Although river discharge increased in the Piedmont Basin after the MSC onset (cf.Natalicchio et al., 2019;Sabino et al., 2020), Bpcren became increasingly 13 C enriched (Fig. 2B).Hence, we infer negligible input also from fluvial Thaumarchaeota to the sedimentary pool and conclude that the measured δ 13 C Bp-cren values reflect the signature of MGI Thaumarchaeota.These values can consequently be used as δ 13 C DIC proxy, despite the moderate to high BIT index (see the Supplemental Material for details on the acyclic and cyclic biphytanes).
The MSC onset coincided with intensification of water-column stratification in the Piedmont and other Mediterranean subbasins (Natalicchio et al., 2017;García-Veigas et al., 2018).This change is mirrored in the studied samples by 13 C enrichment in thaumarchaeal lipids and algal steranes and phytane (see also the Supplemental Material; Fig. 2B).Excursions toward δ 13 C Bp-cren values as high as −15‰ in the proximal Pollenzo sector coincide with [2]/[3] ratios lower than 3 (Figs.2B and 2C).Remarkably, such low ratios are also found in modern sediments of the Mediterranean Sea for which thaumarchaeal lipids are derived dominantly from the upper water column (<200 m; cf.Kim et al., 2015).Accordingly, the pattern in the proximal sector is likely due to lipids sourced from Thaumarchaeota dwelling in the upper water column and fixing carbon from a DIC pool reaching δ 13 C values as high as +5‰ ± 2‰ during the earliest MSC phase.The drier climate in the Mediterranean during the MSC (Roveri et al., 2014) makes preferred 12 CO 2 degassing via enhanced seawater evaporation a possible explanation for such 13 C enrichment (e.g., Horton et al., 2016).However, in the northern Mediterranean, humid conditions persisted (Bertini and Martinetto, 2011), and the climate was only relatively dry during some excursions (cf.Natalicchio et al., 2019;Sabino et al., 2020).Therefore, 12 CO 2 degassing is unlikely to have been the sole mechanism responsible for the positive δ 13 C excursions.Interestingly, similar positive excursions have been reported for the DIC pool of the superficial water of the modern Dead Sea (above the pycnocline; +2.5‰ ≤ δ 13 C DIC ≤ +5‰; cf.Oren et al., 1995).Such δ 13 C excursions are caused by phytoplankton blooms preferen-tially taking up 12 C after episodes of enhanced runoff, which supplied additional nutrients to the basin (Oren et al., 1995).Similarly, periodic increases of riverine runoff and nutrient input were shown to have led to phytoplankton blooms in the Piedmont Basin (Natalicchio et al., 2019).We therefore suggest that massive export of 12 C to the seafloor following productivity pulses resulted in the observed positive excursions in the DIC pool of the upper water column (Fig. 4).Concurrently, stabilization of thermohaline stratification limited the replenishment of remineralized carbon to the upper water column, agreeing with the coinciding 13 C enrichment of algal lipids (Fig. 2B).
In contrast, the MSC sediments of the distal Govone section lack abnormally high δ 13 C Bp-cren values and show [2]/[3] ratios constantly higher than 3 (Fig. 2C).These ratios hint at input of lipids from MGI Thaumarchaeota dwelling in deeper waters (Kim et al., 2015), probably fixing at least partially carbon from the DIC pool below the chemocline.This scenario explains the lower δ 13 C Bp-cren values (∼−21.5‰ on average), the lack of δ 13 C excursions, and the partial mismatch with the trends observed for algal lipids (Fig. 2B).Periodic input of 13 C-depleted DIC into the upper water column is, however, testified by the lowering of the δ 13 C values of algal lipids (Fig. 2B).Given that mixing was suppressed by the stable thermohaline stratifi-cation, fluvial discharge was the most probable source of 13 C-depleted DIC (δ 13 C DIC = −8‰, see above; Fig. 4) at these times.In conclusion, permanent stratification of the water column after the onset of the MSC promoted the development of strong vertical chemical gradients, establishing a 13 C-enriched upper water column overlying a 13 C-depleted water mass in the Piedmont Basin.

SUMMARY AND IMPLICATIONS
The carbon stable isotope signature of crenarchaeol-derived tricyclic biphytane (δ 13 C Bp-cren ) from marine Thaumarchaeota allows us to trace the composition of the pool of DIC in the Piedmont Basin-the northernmost late Miocene Mediterranean subbasin-∼6 m.y.ago at the advent of the Messinian salinity crisis.The water column was typified by a stagnant, deeper body with a δ 13 C DIC of approximately −3.5‰ toward the basin depocenter before and during the advent of the crisis.The onset of the event was marked by significant 13 C enrichment of the DIC pool in the upper water column (δ 13 C DIC as high as ∼+5‰) due to the basinwide stabilization of thermohaline stratification.The 13 C enrichment is interpreted to primarily reflect preferential export of 12 C to the seafloor after phytoplankton blooms and limited replenishment of remineralized DIC.

Figure
Figure 4. δ 13 C DIC values in stratified water mass of the Piedmont Basin at the advent of the Messinian salinity crisis.DIC-dissolved inorganic carbon.