Comment on cp-2020-163 Anonymous Referee # 1 Referee comment on " Co-evolution of terrestrial and aquatic ecosystem structure with hydrological change in the Holocene Baltic Sea

This is a well-written manuscript that presents analyses of biomarkers and stable isotopes of specific compounds. The analyses are state of the art and have not previously been used in the south-western Baltic. It is important to use new methods to improve our understanding of past environmental changes. However, the interpretations of the data are not straightforward and after reading the manuscript I did not feel that I learned much new about the history of the Baltic Sea. I am sorry but I don’t feel that the study “provided new insights into changes ... in the vegetation in the western Baltic region throughout the Holocene” (line 210).

. Shorter-chain n-alkanes (< C21) are principally produced by phytoplankton and bacteria (Meyers and Ishiwatari, 1993;Zhang and Sachs, 2007), and their H isotopic composition also correlates strongly with growth water (Sachse et al., 2004). Moreover, n-alkanes can be transported to sediments as aerosolized particles that are deposited directly in coastal waters and lakes or transported by rivers (Schefuss et al., 2005). The spatial coverage of this atmospheric 70 transfer is dependent upon climate conditions and density of vegetation, but n-alkanes are generally deposited within weeks after being aerosolized (e.g., Nelson et al., 2018), making them ideal for paleoenvironmental reconstructions.

Glycerol dialkyl glycerol tetraethers (GDGTs)
For GDGT analyses, an second aliquot of the polar fraction -at a concentration of 2 mg mL -1 -was dissolved in hexane : isopropanol (IPA) 99:1 (v / v) and analyzed using ultra high performance liquid chromatography mass spectrometry on an Agilent 1260 UHPLC coupled to a 6130 Agilent MSD following Hopmans et al. (2016). The Branched and Isoprenoid Tetraether (BIT) index (Hopmans et al., 2004) was calculated as follows: 135 where roman numerals refer to the number of methyl groups attached to the 5 or 5' positions of the alkyl chain (zero, one, and two, respectively). GDGT concentrations were calculated based on a known concentration of the C46 GDGT standard (Huguet et al., 2006).
The #ringtetra, used to assess the source of brGDGT in the marine water column, where values higher than 0.7 indicate 140 sedimentary in situ production (Sinninghe Damsté, 2016) was also used. #ringtetra is defined as follows: The roman numeral I refers to the number of methyl groups, as indicated for the BIT index. The letters 'a ', 'b', and 'c' refer to the number of cyclopentane moieties (zero, one, and two, respectively).

Compound-specific isotope analyses 145
Compound-specific carbon isotope analyses of n-alkanes (δ 13 Calkane) were performed in duplicate on a Thermo Trace GC (1310) coupled with a Thermo Delta-V plus IRMS at the Climate Geology Department of the ETH Zurich. The GC was equipped with a RTX-200 60 m capillary column (Restek, 60 m × 0.25 mm × 0.25 μm). The temperature program was as follows: ramp from 40 °C to 120 °C at 40 °C min -1 , followed by a 6 °C min -1 ramp to 320 °C, held at 320 °C for 12 min.
Hydrogen isotope ratios of n-alkanes (δ 2 Halkane) were measured at the Royal Netherland Institute for Sea Research in duplicate 150 by GC coupled to an IRMS via a high temperature conversion reactor. The GC was equipped with a CP Sil-5 column (Agilent, 30 m × 0.25 mm × 0.25 μm). The temperature program was as follows: started at 70 °C, increased to 130 °C at 20 °C min -1 , increased to 320 °C at 4 °C min -1 , held at 320 °C for 10 min. At the start of each day, the H3 + factor was measured and corrected for (Sessions et al., 2001). H3 + values ranged from 4.3 to 4.6. Prior to measurement of samples for both C and H isotopes, a nalkane mixture (Mix B, supplied by A. Schimmelmann, Indiana University) was measured. Sample analyses were only conducted when the average difference and standard deviation between online and offline values was less than 5‰ for H and 0.5‰ for C. For both C and H isotope measurements, the reported standard error is based on duplicate analyses.

Results
The core covers the last 10.6 ka with average sedimentation rates between 55 and 250 cm ky -1 (Weiss et al., 2020). The large 160 variations in sedimentation rates for core 64PE410_S7 and the nearby cores to which it has been correlated are likely related to the shallow water depth in the Arkona Basin (~ 45 m) as well as changes in connection with the North Sea. Bulk isotopes (δ 13 Cbulk and δ 15 Nbulk values, as well as TOC and TN content), lipid concentrations, and compound-specific n-alkane isotope analyses were conducted on 22 samples.

Bulk isotopes 165
TOC and TN ranged from 0.5 to 7.2 and 0.1 to 0.8, respectively, with higher values in the most recent part of the core (Fig.   S1). δ 13 Cbulk values varied by ~3‰ in the Early Holocene (< 7.7 ka) followed by a small shift after the marine transgression ( Fig. S1). δ 15 Nbulk values were elevated at the transition from the YS to the AL (5.5‰) followed by a drop to 2.8‰ in the early AL phase (10.1 ka). Values reached 4‰ during the latter half of the AL, succeeded by a second drop at the marine transgression (2.8‰). Values slowly increased during the early LS phase. Values of 4 ± 1‰ occurred across the MB phase (Fig. S1). 170 Elemental C and N (C/N) ratios ranged from 5.6 to 13.4, spanning common values for both marine and terrestrial ecosystems (Thornton and McManus., 1994). C/N ratios are plotted against δ 13 Cbulk values for all samples; a mix of freshwater, marine, and terrestrial C3 plant-derived values are recorded, which group by established Baltic Sea phases (Fig. 2).

n-alkane distributions and isotopic composition
Organic carbon-normalized concentrations of n-alkanes (C21 to C35) were highest in the older part of the record (2.8 µg gTOC -1 175 at 10.6 ka, Fig. 3), followed by a decrease until 9.4 ka (with a notably low concentration at 10.2 ka). The relative abundance of n-alkane homologues is comparatively invariant across the record (Fig. 3), except for a slight increase in the abundance of the C21 n-alkane following the marine transgression and absence or low abundance of the C17 n-alkane during the AL. The most abundant n-alkane is the C29 (21 ± 4 %, n=23), followed by the C27 (19 ± 2 %, n=23), C25 (16 ± 3 %, n=23) and C31 (16 ± 3 %, n=23). C and H isotope values are plotted as weighted averages for short-chain (C21), mid-chain (C23 to C25), and long-180 chain (C27 to C31). Both δ 13 Calkane and δ 2 Halkane values captured substantial fluctuations during the early part of the record (before ca. 7.7 ka), but show relatively stable signals for both following the marine transgression, likely the result of low sampling resolution (Tables 2 and 3; Figs. 4 and 5). Weighted-average δ 13 C values of the mid-and long-chain n-alkanes were similar from 10.6 to 8 ka, after which the mid-chain n-alkanes shifted toward higher values and the long-chain n-alkanes remained similar (Fig. 4) Values varied from 2.0 ± 0.7 ‰ between 11 -7.7 ka to 6.7 ± 1.4 ‰ between 7.3 -1.3 ka (Fig. 6a). H isotope values of short-, mid-, and long-chain n-alkanes tracked the same trends across the record. The δ 2 H values of all n-alkanes were higher in the oldest part of the record (YS, prior to 10.6 ka), and recorded a rapid decrease of ~50‰ at the onset of the AL phase (Fig. 5). 190 Subsequently, a second, considerable increase of ~50‰ occurred between 10 ka and 9.2 ka, succeeded by a gradual decrease lasting until the marine transgression at 7.7 ka. δ 2 H values showed higher values across the marine transgression. Data is limited for the LS phase, but no large isotopic shifts were noted in the samples analyzed here.

Abundance of LCDs and BIT index
Low concentrations of LCDs were detected in a majority of samples prior to the marine transgression. Furthermore, no 1,12-195 diols were detected in the core indicating limited input from Proboscia diatoms (de Bar et al., 2020). The C28 1,14-diol was only present during the MB phase. For all other LCDs, the main change in abundance (relative to all LCDs) occurred at 7.9 ka with a drop in FC32 1,15-diol (F = fractional abundance, from 20 to 1%) and an increase in FC30 1,15-diol (30 to 80%). LDI-temperature reconstructions (Table S1) were similar to those of Kotthoff et al. (2017) with relatively high temperatures during the YS (18.5 ± 0.2 ⁰C) and a decrease during the AL (11.5 ± 2.4 ⁰C). Maximum temperatures occurred during the LS phase, in agreement 200 with the Holocene thermal maximum in Europe (21.5 ± 1.5 ⁰C, 24 ⁰C at 7.2 ka). Temperatures during the MB were similar to modern summer temperatures (17.2 ± 0.1 ⁰C). The coldest period (AL) is also the period with the highest proportion of C32 1,15-diol.
The distribution of brGDGTs changed drastically across phases ( Fig. S2) with a distribution similar to soils for all phases except the AL, which suggests mixing between lacustrine and soil or permafrost-derived brGDGTs. BIT values were higher 205 in the older part of the record (0.8 before 9.9 ka), then continuously decreased to 0.1 at 6.5 ka. Similar BIT values were calculated for all LS samples (Fig. 6). #ringtetra was lower than 0.7 before the transgression (0.5 ± 0.1). A rapid increase occurred at the transgression (~7.7 ka) and values remained consistently high afterwards (0.7 ± 0.1, Table S2).

Discussion
Compilation of biomarker distributions with corresponding n-alkane C and H isotope compositions provided new insights into 210 changes in the regional hydrology and vegetation in the western Baltic region throughout the Holocene. Isotope and biomarker data are discussed below by Baltic Sea phase.

Yoldia Sea
The record covers only a brief snapshot of the end of the YS phase, a period in Baltic Sea history thought to have higher salinity as evidenced by the presence of marine phytoplankton species (Sohlenius et al., 1996). The presence of short-chain et al., 1970;Meyers and Ishiwatari, 1993). The only LCDs present are the C30 1,13-, C30 1,15-, and the C32 1,15-diols. LCDs are produced by marine and freshwater eustigmatophyte species (Balzano et al., 2018;Rampen et al., 2014;Volkman et al., 1992). The presence of the C32 1,15-diol, produced by eustigmatophyte species in rivers and stagnant freshwater pools (e.g., Lattaud et al., 2017;Balzano et al., 2018), implies low salinity to freshwater conditions. Corresponding δ 13 Cbulk values versus 220 C/N ratios for this phase fall just on the edge of values common for freshwater ecosystems (Fig. 2), and along with the presence of the C32 1,15-diol, suggest that salinity of the basin may have already significantly declined by the end of the YS phase. In fact, some records divide the YS into two parts: an early marine phase followed by a freshwater phase (e.g., Sohlenius et al., 1996). The highest n-alkane concentrations of the whole record, together with highest δ 2 Halkane values, occur just at the phase boundary ( Fig. 3 and 5). The increase in δ 2 Halkane values varies from around 12‰ (C21 and C23 n-alkanes) to 47‰ (C29 n-225 alkane), and may be linked to regional climate conditions -likely increased warming and meltwater -which drove the shift from the YS phase into the low salinity AL phase.

Ancylus Lake
The AL phase was the most dynamic of the whole record and includes a number of large isotopic shifts. C isotope compositions of mid-and long-chain n-alkanes follow similar trends during the AL. A series of C isotope fluctuations of around 2‰ were 230 recorded during the AL. There are two shifts to more negative values: one at 10.2 ka and one at 8.2 ka, and an increase at 9.2 ka. The n-alkanes and their C isotope signatures imply largely terrestrial, higher plant input during the AL, supported by the absence of the aquatically produced C17 n-alkane during most of this phase (Fig. 3). The LEWIS index values are the lowest of the record (Fig. 6a), suggesting a lack of species diversity (Magill et al., 2019). This is supported by nearby pollen records that indicate the same absence of higher plant diversification, likely due to the relatively cold temperatures in Northern Europe 235 at this time (Seppä and Birks, 2001;Seppä et al., 2005;Antonssson et al., 2006), which explains the similarity of n-alkane C isotope values. In contrast, the δ 13 C values of C21 n-alkane were higher compared with the longer-chain (> C27) n-alkanes (-29 versus -21‰ before 7.8 ka), suggestive of mixed terrestrial and aquatic production or emergent aquatic productivity (Ficken et al., 2000).

Ancylus Regression 240
At 10.2 ka, the Baltic Sea experienced a large regression (Moros et al., 2002), a likely reflection of continental uplift that resulted in shallowing and freshening of the basin. C isotope values of all n-alkanes record a decrease at the time of the AL regression and LEWIS index values are low, suggesting low diversity. Low concentrations of n-alkanes (~150 ng gTOC -1 , Fig. 3) occurred at the AL regression, implying less continental runoff into the Arkona Basin at this time. Simultaneously, there was a 15 -20‰ increase in δ 2 Halkane values (Fig. 5). Following the Ancylus Regression, δ 2 Halkane values become lower, 245 coincident with a rapid, order of magnitude increase in concentration of n-alkanes ( Fig. 3 and 5). The SIS was retreating at this time (Muschitiello et al., 2015;Cuzzone et al., 2016), thus it is plausible that a meltwater pulse transported a higher concentration of n-alkanes from the north into the basin just after 10.2 ka. A coincident increase in the BIT index from 0.5 to https://doi.org/10.5194/cp-2020-163 Preprint. Discussion started: 12 January 2021 c Author(s) 2021. CC BY 4.0 License. 0.8 (Fig. 6b) and #ringtetra values below 0.7 (suggesting a lack of in situ production, Sinninghe Damsté, 2016), strengthen the argument for increased input of allochthonous material into the basin. The higher proportion of GDGT-III (Table S2) is 250 indicative of combined in situ brGDGT production and permafrost or soil erosion (Russell et al., 2018;Warden et al., 2018;Kusch et al., 2019). No major changes appear to have occurred in the water column, but progressive melting of the SIS likely caused transport of soil brGDGTs leading to the increase in BIT index values. Furthermore, a peak in FC32 1,15-diol to around

Ancylus Lake vegetation and hydrological change
Following the Ancylus Regression, there were two shifts in mid-and long-chain n-alkane C isotope signatures: an increase (+2‰) around 9.2 ka, followed by a return to lower values, and a second increase (again +2‰) at the onset of the marine transgression (Fig. 4). These shifts in δ 13 Calkane values during the AL phase might reflect the advance and retreat of coniferous cover as conifers, such as Pinus and Juniperus, have higher δ 13 Calkane values compared with angiosperms (Diefendorf and 265 Freimuth, 2017). Since Pinaceae produce low concentrations of n-alkanes compared to Juniperus (Diefendorf et al., 2011;Lane, 2017), these C isotope shifts can be tentatively attributed to Juniperus shrub extension. These isotopic shifts align with the main climatic changes recorded in Swedish and Finnish lakes (Seppä and Birks, 2001;Seppä et al., 2005;Antonssson et al., 2006). The maximum extension of Pinus and Juniperus was recorded at 9.2 ka in these regional lakes (Seppä and Birks, 2001;Seppä et al., 2005;Antonsson and Seppä, 2007), coincident with elevated δ 13 Calkane values. Similarly, a large increase in 270 the angiosperm Alnus (which tends to have more lower δ 13 Calkane values) was observed at 8.7 ka at the time of a decrease in Juniperus cover north of the Baltic Sea (Antonsson and Seppä, 2007), paralleling the return to lower δ 13 C values. A significant increase in δ 2 Halkane values (~ 50‰) began after 10 ka and peaked at 9.2 ka (Fig. 5), precisely at the time when a similar increase in δ 2 H values of haptophyte-derived alkenones from the same core was noted (Weiss et al., 2020). The H isotope composition of terrestrial plants reflects local meteoric water signals (Sachse et al., 2012), and is thus subject to large 275 fluctuations resulting from variations in the hydrological cycle. H isotope values of precipitation were reconstructed from C23 and C29 n-alkanes using linear models from McFarlin et al. (2019), and range from -125‰ to -62‰ (residual standard error = 14) and -107‰ to -36‰ (residual standard error = 15), respectively, across the AL phase (Fig. 5). The lowest δ 2 Halkane values -and in turn δ 2 H values of reconstructed precipitation -occur during or immediately preceding the 50‰ increase (Fig. 5).
influence from an isotopically lighter (more negative) water source affected isotopic signatures of aquatically-produced nalkanes. Model data suggests that the H isotope composition of the SIS was around -310‰ (De Boer et al., 2014), and modern mean annual precipitation in the region is -63‰ (Bowen and Revenaugh, 2003), highlighting the marked isotopic offset between the two sources. Larger volumes of meltwater from the SIS would result in significantly more negative δ 2 H values for aquatically-derived mid-chain n-alkanes, in line with what is observed here at around 10 ka (Fig. 5) and also recorded by 285 haptophyte algae-derived long-chain alkenones from the same core (Weiss et al., 2020). After 10 ka the decrease of melt water and increase in precipitation (Dahl and Nesje, 1996;Seppä and Birks, 2001;Hammarlund et al., 2003) likely caused the dominant water source to become isotopically heavier, resulting in increased n-alkane H isotope values.
The global transition from a glacial into an interglacial climate state across the Holocene, was punctuated by a few abrupt cold events observed in Northern Hemisphere records (e.g., Fleitmann et al., 2008), and in glacier and ice sheet oscillations. At 9.2 290 ka, the peak in δ 2 Halkane values, there was a slowdown of thermohaline circulation due to a large meltwater pulse from the Laurentide Ice Sheet into the North Atlantic, causing a brief cold event in the Northern Hemisphere (Fleitmann et al., 2008).
While our record is of insufficient resolution to capture this rapid event, the increase of δ 2 Halkane values noted at 9.2 ka is presumably also influenced by the environmental conditions present at that time. Another rapid cold event occurred at 8.2 ka, which is not observed in our record, but may be elucidated with higher resolution sampling at this time interval. 295

Marine Transgression
A large marine transgression re-established the connection between the Baltic Sea and the North Sea at around 7.7 ka (Moros et al., 2002). At the onset of the transgression (which lasted from 7.7 to 7.2 ka), long-chain n-alkane (> C27) concentrations increased and the shorter-chain n-alkanes are detected (< C19, Fig. 3), indicating greater input from terrestrial and algal sources, respectively. Regional warming began just prior to this period (Seppä and Birks, 2001;Seppä et al., 2005;Antonsson and 300 Seppä, 2007), which favored the growth and diversification of terrestrial and aquatic plants. At the start of the transgression, mid-and long-chain n-alkane C isotope values began to diverge (LEWIS index increases, Fig. 6b), with the mid-chain nalkanes becoming more positive by about 4‰ (Fig. 4). The δ 13 Calkane values of long-chain n-alkanes showed a C3 higher plant signal (-30.4 ± 0.7‰), while the mid-chain n-alkanes were higher (-27.7 ± 0.4‰), characteristic of submerged aquatic plants (Ficken et al., 2000). The C21 n-alkane, produced by submerged aquatic plants and freshwater and marine phytoplankton, was 305 the highest prior to the transgression, and became lower by ~5‰ across the transgression, despite the fact that large volumes of saline water entered the basin and the δ 13 Cbulk values showed increased values. Terrestrial vegetation is general relatively 13 C-depleted compared to marine organisms (Fig. 2), so the more negative values noted for the C21 n-alkane might indicate a relative increase in the abundance of submerged aquatic plants and freshwater phytoplankton rather than the influx of saline water. An increase in δ 2 Halkane values also occurred across the transgression (except the C23 n-alkane which became more 310 negative). The C23 n-alkane is known to be produced by Sphagnum species (Baas et al., 2000;Vonk and Gustafsson, 2009), and thus may be reflecting a different water source than the rest of the n-alkanes. Potential alternative water sources for https://doi.org/10.5194/cp-2020-163 Preprint. Discussion started: 12 January 2021 c Author(s) 2021. CC BY 4.0 License.
Sphagnum are the stagnant water of meltwater ponds which would have very negative H isotope values. TOC and δ 13 Cbulk values increased, while C/N ratios decreased, likely due to enhanced phytoplankton production (cyanobacterial blooms in particular, Bianchi et al., 2000), and amplified input of organic matter into the sediments from increased erosion and primary 315 production (Sohlenius et al., 2001). The BIT index continued to decrease (Fig. 6b), while #ringtetra increased (> 0.7) indicating a change in the main brGDGT producers from (freshwater) lacustrine production towards (marine) sedimentary pore-water production. The decrease in BIT values at the transgression may be the result of a shift in freshwater versus marine producers rather than a decrease in soil-input. A rapid decrease in FC32 1,15-diol occurred from the end of the AL phase into the marine transgression (8.0 to 7.3 ka; Fig. 6b). The decrease may be the result of increased salinity in the basin which likely caused a 320 change in the LCD-producing population from freshwater to brackish and marine LCD-producing species known to synthesize less of the C32 1,15-diol.

Littorina Sea and Modern Baltic phases
The transition from the marine transgression into the early half of the Littorina Sea (LS) phase is characterized by variable δ 2 Halkane and δ 13 Calkane values (Figs. 4 and 5). Multiple studies (e.g., Hammarlund et al., 2003;Seppä et al., 2005;Antonssson 325 et al., 2006;Antonsson and Seppä, 2007) indicate that the period between 8.0 and 4.2 ka was warm, dry, and stable, followed by a period of rapid hydrological changes. This is supported by the LDI-reconstructed summer sea surface temperature (Table   S1), indicating an optimum between 7.3 and 5.3 ka (LS average of 21.5 ± 1.5 ⁰C). In addition, stable #ringtetra, BIT, and FC32 1,15-diol suggest relatively unchanged bacterial communities and river inflow. However, the C and H isotope data suggest instability and diversification in the catchment from the marine transgression until the middle of the LS phase. We note, 330 however, that the rapid hydrological changes evidenced in other records and the instability suggested by isotopic data discussed here likely do not capture the nuances of the time period as a result of low sampling resolution. At the transition from the LS into the Modern Baltic phase, the C28 1,14-diol, characteristic of Proboscia diatoms (Rampen et al., 2007) and Apedinella radians (algae mainly found in estuaries, Rampen et al., 2011) is present in relatively high amounts. This aligns with data from Andrén et al. (2000), which indicated that freshwater diatoms were low throughout the LS phase, but were more prominent 335 during the Modern Baltic phase.

Conclusions
The Holocene history of the Baltic Sea consisted of variations in regional vegetation and hydrology that were connected to global climate phenomena. The Ancylus Lake phase, in particular, was characterized by large fluctuations in the extent of 340 gymnosperm cover correlated with the gradual warming in the catchment and substantial shifts in source water from a significantly light, meltwater-influenced source to an isotopically heavier precipitation-dominated source. These findings align with a previous study which found a similar source water shift over the same time interval. Subsequent large meltwater pulses https://doi.org/10.5194/cp-2020-163 Preprint. Discussion started: 12 January 2021 c Author(s) 2021. CC BY 4.0 License. from the Laurentide Ice Sheet, which caused a slowdown of thermohaline circulation that triggered two rapid, regional cold events at 9.2 and 8.2 ka, likely contributed to the lack of diversification of terrestrial vegetation noted for this period. The 345 marine transgression that ended the low salinity Ancylus Lake phase was accompanied by a diversification of terrestrial higher plants as well as changes in coastal aquatic ecosystems as a result of regional warming which continued into the Late Holocene.
Our record showed an offset between previously established phase boundaries based on salinity and eustatic changes and the fluctuations in hydrology and higher plant diversification. Proxy signals like the record discussed here highlight the complex temporal dynamics at play between paleo archives. For example, shoreline changes can be temporally offset from riverine 350 transport of plant waxes. Use of multiple proxies is essential to fully understand the complexity of paleoenvironments, but spatial and temporal resolution of individual proxies must be acknowledged.

Author contribution
GW and JL designed the study, extracted samples, conducted isotopic analyses, and contributed equally to writing of the manuscript with contributions from all co-authors. 355