Group 2i Isochrysidales produce characteristic alkenones reflecting sea ice distribution

Alkenones are biomarkers produced solely by algae in the order Isochrysidales that have been used to reconstruct sea surface temperature (SST) since the 1980s. However, alkenone-based SST reconstructions in the northern high latitude oceans show significant bias towards warmer temperatures in core-tops, diverge from other SST proxies in down core records, and are often accompanied by anomalously high relative abundance of the C37 tetra-unsaturated methyl alkenone (%C37:4). Elevated %C37:4 is widely interpreted as an indicator of low sea surface salinity from polar water masses, but its biological source has thus far remained elusive. Here we identify a lineage of Isochrysidales that is responsible for elevated C37:4 methyl alkenone in the northern high latitude oceans through next-generation sequencing and lab-culture experiments. This Isochrysidales lineage co-occurs widely with sea ice in marine environments and is distinct from other known marine alkenone-producers, namely Emiliania huxleyi and Gephyrocapsa oceanica. More importantly, the %C37:4 in seawater filtered particulate organic matter and surface sediments is significantly correlated with annual mean sea ice concentrations. In sediment cores from the Svalbard region, the %C37:4 concentration aligns with the Greenland temperature record and other qualitative regional sea ice records spanning the past 14 kyrs, reflecting sea ice concentrations quantitatively. Our findings imply that %C37:4 is a powerful proxy for reconstructing sea ice conditions in the high latitude oceans on thousand- and, potentially, on million-year timescales.

However, application of U K0 37 -SST calibrations in the northern high latitude oceans has encountered major difficulties. Variable warm bias in U K0 37 -based SST reconstructions is often accompanied by the occurrence of tetra-unsaturated alkenone (C 37:4 ), which is usually absent in mid-to-low latitude oceans and E. huxleyi cultures even under exceedingly low growth temperature 8 . This pattern is reported in surface sediment and seawater filtered particulate organic matter (POM) samples from the Nordic Seas and the Bering Sea [14][15][16] , and downcore sediment records in Okhotsk Sea during glacial periods [17][18][19] . As a result, Rosell-Melé 14 concluded that U K0 37 was unreliable when the percentage of C 37:4 among the total C 37 methyl alkenones (%C 37:4 ) is >5%. Yet, %C 37:4 greater than 5% is widely reported in sediment records in the northern high latitude oceans, thereby complicating SST reconstructions from these regions ( Supplementary  Fig. 1).
On the other hand, negative correlations between %C 37:4 and sea surface salinity (SSS) on a regional scale were observed 14,15,20 , with elevated %C 37:4 often associated with fresher and colder water mass 21,22 . The mechanisms for these observations, however, are poorly defined, with previous studies suggesting unknown oceanographic parameters 14 and/or alkenone producers 22 as the possible explanations. The occurrence of miscellaneous compounds that co-elute with C 37:4 alkenone in sediment samples extracts adds an additional level of complication when interpreting %C 37:4 data 23 . Regardless of these problems, elevated %C 37:4 is widely applied as an indicator of fresher and colder polar water mass or decreased SSS (Supplementary Table 1). More recently, it has been applied as an indicator of sea ice margin based on co-occurrence with benthic foraminifera signal suggesting sea ice breakup 24 . The interpretation of %C 37:4 as a proxy for SSS stands in direct contrast to results from culture experiments, which show positive or no correlation between salinity and %C 37:4 in marine and brackish Isochrysidales species [25][26][27] . Such conflict suggests that elevated %C 37:4 is not a physiological response to decreased SSS, but rather that unidentified producers of C 37:4 occur in high latitude marine environments 14,22 .
In this study, we used next-generation sequencing (NGS) to identify Isochrysidales in water column, sea ice, and sediment samples in northern high latitude marine environments. We show that Isochrysidales communities in subarctic regions are composed of a lineage that is distinct from E. huxleyi and any other described species. Culture of this lineage isolated from sea ice demonstrates that they are prolific producers of C 37:4 . %C 37:4 in POM and surface sediments in northern high latitude oceans show strong correlation with annual mean sea ice concentrations, which is further corroborated by downcore sediment records from the Svalbard region spanning the past 14 kyr. Our results suggest that %C 37:4 has great potential as a paleo sea ice proxy, which may provide crucial information about sea ice distributions prior to satellite observations.

Results and discussion
Identifying widespread ice-related alkenone producers. We sequenced the V4 region of the 18S rRNA gene from three surface sediment samples containing high %C 37:4 (59-68%; Supplementary Fig. 2, Supplementary Data 1) that were collected from Victoria Strait in the Canadian Arctic Archipelago where the sea ice regime is mainly governed by first-year ice. Four Isochrysidales amplicon sequence variants (ASVs) were recovered from those surface sediments (Supplementary Table 2). We also extracted 18S-V4 sequences of Isochrysidales available from NGS datasets from high latitude oceans (20 datasets, 1216 samples; Supplementary Data 2). We used a phylogenetic framework to compare these data with published Isochrysidales 18S-V4 data from environmental and culture studies 28 (Fig. 1).
We followed the Isochrysidales classification of Theroux et al. 29 based on habitat salinity ranges and phylogenetic divergence: Group 1 (freshwater species, uncultured), Group 2 (brackish species, e.g., Isochrysis galbana, Ruttnera lamellosa), and Group 3 (marine species, E. huxleyi and G. oceanica). Our phylogenetic analysis revealed close relationships between the majority of Isochrysidales sequences recovered from marine sites with perennial or extended seasonal sea ice and those of published sequences from lakes with seasonal or perennial ice cover (Figs. 1 and 2a). The sequences formed a monophyletic group with 80% bootstrap support, and placement of these sequences within Group 2 Isochrysidales is supported by 100% bootstrap support. We refer to this monophyletic clade as the Group 2 ice lineage Isochrysidales (Group 2i). Group 2i is distinct from any named Isochrysidales species, and potentially represents one or more new species. The sequences falling within Group 2i were detected in environments with a wide range of salinity, from freshwater to saline brine channels within sea ice, and in samples from sea ice cores, under ice seawater, and sediments from regions governed by multi-and first-year ice. These distributions suggest that Group 2i is euryhaline and well-adapted to variable Arctic marine environments. E. huxleyi was abundant in seawater samples that were collected during the summer after ice-melt in the Chukchi Sea and Barents Sea, and was not identified in any of the sea ice samples except one sample from the central Arctic. Particularly, seawater samples collected in the Fram Strait during sea ice melting season (June 2014) 30 where the western part of the Fram Strait was ice-covered and eastern part was ice-free showed E. huxleyi was present in both ice-covered and ice-free sites. However, Group 2i was only detected in seawater samples collected under ice cover. The DNA data suggest that the presence of Group 2i is tightly associated with sea ice.
Culture experiments show Group 2i produce predominant C 37:4 . To confirm that Group 2i produces high %C 37:4 , we cultured the unclassified Isochrysidales strain RCC5486 at 3 and 6°C in f/2 medium under salinity of 15, 21, 26, 31, and 38 ppt. The RCC5486 strain falls within the Group 2i in our phylogeny and it is the most phylogenetically similar strain to the Isochrysidales found in Canadian Arctic Archipelago surface sediments that is available in pure culture (Fig. 1). Live cells of RCC5486 were isolated during the algal pre bloom and bloom-development phase in sea ice samples from Baffin Bay 31 (67.47°N, 63.78°W; May-July 2016). The alkenones produced by RCC5486 showed exceptionally high %C 37:4 (~80%) at both 3 and 6°C ( Supplementary Fig. 2), and salinity does not have any significant influence on %C 37:4 (Supplementary Fig. 3). This further validates that physiological response to decreased salinity would not result in increased C 37:4 production. Published DNA sequences that fall into Group 2i in our phylogenetic analyses were also observed in water and sediment samples from lakes in China 29,32 , Canada 33 , the USA, 29 and Antarctica 34 , where C 37:4 was the predominant alkenone. High %C 37:4 alkenone production is thus likely a shared characteristic across Group 2i Isochrysidales. Culture of RCC5486 also showed an absence of C 38 methyl alkenones (C 38 Me), matching alkenone production by other Group 2 Isochrysidales and differing from E. huxleyi 8 . However, C 38 Me was present in the Canadian Arctic Archipelago surface sediments, even though Group 2i was the only Isochrysidales detected through NGS. The C 38 Me detected in Canadian Arctic Archipelago is likely produced by E. huxleyi even though their DNA was not detected. The discrepancy between DNA and alkenone preservation could result from differences in the life cycles of Group 2i compared to E. huxleyi: the former potentially forms cysts and enters a resting stage in surface sediment, 35,36 while the latter does not form resting spores during winter 37 , after the bloom they die and disaggregate during downward transportation into sea floor 38 .
Implications for sea ice, SSS, and SST reconstructions. The identification of the novel and widespread Group 2i Isochrysidales gives rise to the possibility of using %C 37:4 as a reliable sea ice proxy. Elevated %C 37:4 from surface sediment and seawater POM 15,22 is located at regions with seasonal sea ice in the Northern Hemisphere (Fig. 2b). %C 37:4 in both POM and surface sediment were positively and significantly correlated with sea ice concentrations (Fig. 3a, c). Differences in the calibration could be results of stronger seasonality of POM, uncertainty in the age of the surface sediment, and advection influencing the linkage between sediment and overlying surface water 12 . The positive correlation between sea ice concentration and %C 37:4 most likely reflect increasing alkenone contribution from Group 2i Isochrysidales with increasing sea ice. %C 37:4 showed negative and significant correlations with SSS in POM and surface sediments (Fig. 3b, d), consistent with prior research 14,15 . However, this relationship is a byproduct of the negative correlation between SSS and sea ice concentrations in the Maximum-likelihood tree based on 18S rRNA gene from 99 sequences spanning three groups of Isochrysidales and 6 from the Coccolithales outgroup. The numbers shown on branches represent bootstrap support for the node (only those >50% are shown). Numbers in the parentheses represent numbers of sequences included in the collapsed groups, and the expanded version of this tree can be found in Supplementary Fig. 4. The scale bar represents substitution/site. Sequences generated from Canadian Arctic Archipelago surface sediments in this study are highlighted in red, and sea ice isolated strain RCC5486 cultured in this study is highlighted in blue. Sequences from reanalyzed NGS datasets are highlighted in brown, and other sequences were obtained from NCBI GenBank. The branches of the ice lineage are marked in red. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-20187-z ARTICLE above sample sites, which reflects the temporal influence of melting sea ice on near-surface salinity ( Supplementary Fig. 5). Thus variations in %C 37:4 cannot be attributed to SSS changes, and regional relationship between %C 37:4 and SSS should not be extrapolated for global-scale SSS reconstruction. So far there is no mechanistic explanation that supports a direct response of %C 37:4 to changes in SSS, except in low salinity (0-6 ppt) or estuarine environments where Group 1 Isochrysidales are present and contribute to elevated %C 37:4 29,39,40 . Group 1 Isochrysidales are typically found in freshwater to oligosaline environments and predominantly produce C 37:4 41 . Alkenone contribution from Group 1 can be identified through the presence of C 37:3b isomer, which is not produced by Groups 2 and 3 41 . Our results show that both Group 1 and the Group 2i are present in brackish water underlying sea ice in the northwest of Gulf of Finland, Baltic Sea 42 ( Supplementary Fig. 6). However, Group 1 Isochrysidales are very sparse within sea ice in the Baltic Sea and are absent in other sea ice samples examined in this study (Fig. 2). Thus, we conclude that the majority of C 37:4 production in the northern high latitude oceans distal from coast can be attributed to the presence of sea ice and Group 2i Isochrysidales.
Alkenones produced by the Group 2i are also a potential source of bias in U K0 37 -SST reconstructions. Surface sediment samples from regions covered by winter sea ice in the North Atlantic experience significant warm bias in U K0 37 -SST 13,43 . A similar warm bias is also observed in calculated SSTs from surface sediments examined in this study, including the Canadian Arctic Archipelago samples where Group 2i was detected (Fig. 2c); however, the exact mechanism leading to the warm bias is still a question. Group 2i may produce alkenones within multiyear sea ice, but there is no prior report of alkenones detected from the surface sediments in the central Arctic. Bloom of Group 2i has been observed upon ice-melt in Lake George, USA, followed by a bloom of non-ice lineage Isochrysidales 44 . Group 2i and other Group 2 Isochrysidales are able to form cysts in sediments and potentially re-emerge to surface water during water-overturning under increased insolation 35,36 , which could occur in blooms during ice-melt. Prolonged seasonal sea ice and the Group 2i Isochrysidales within sea ice could potentially shift the phenology of E. huxleyi from colder to warmer seasons. They are also likely to have a U K0 37 -SST calibration different from E. huxleyi. The bias in U K0 37 -SST introduced by Group 2i could thus be more prominent during periods with extended seasonal sea ice coverage in downcore records. More experimental and field studies are needed to fully understand the causes for the warm SST bias inferred by U K0 37 in high latitude oceans.

kyr (Figs. 4 and 5)
. IP 25 is a C 25 isoprenoid lipid synthesized by certain Arctic sea ice diatoms and is deposited in underlying sediments following ice-melt in spring [53][54][55] . Coarse sediment fraction at this site is largely composed of lithic grains and foraminifera, reflecting sea ice formation off the western Barents shelf 56 . The Fram Strait is the major gateway between the North Atlantic and the Arctic Ocean, and sea ice distribution in our study area is highly sensitive to surface temperature variability in modern time 57 . Over longer timeframes, such as the Holocene, variations in sea ice extent in the Fram strait, the Greenland Sea, and the northern Barents Sea co-occurred with changes in Northern Hemisphere solar insolation and changes in Greenland air temperature [58][59][60][61][62] . Marine sediment core M23258-2 56,63 (74.99°N, 13.97°E) is located close to the modern maximum winter sea ice extent in the northeastern Norwegian Sea where temporal variations of sea ice conditions have been identified 64 . Alkenones were not detected prior to 14.1 kyr BP in M23258-2, likely due to seasonally extended sea ice cover limiting productivity. The general trend of %C 37:4 from M23258-2 63 is in direct agreement with inferred-temperature trends from the δ 18 O values of Renland ice core located in east Greenland 65 , with increasing %C 37:4 corresponding to decreasing temperature, and is positively correlated with IP 25 concentration and coarse fraction content in M23258-2 ( Supplementary Fig. 7). The high values of %C 37:4 in both M23258-2 and JM09-020-GC 52    Hemisphere, as the coarse fraction contents in this core also increased, reflecting sediment injections caused by cascades of dense brine water down the Barents shelf induced by seasonal sea ice 56 . IP 25 in M23258-2 is, however, absent (or below detection limit) during this time period. The return to partial seasonal sea ice cover during neoglaciation is also evident in the rapid IP 25 increase in the Fram Strait 60 (MSM5/5-723-2; 79.16°N, 5.33°E) and northern Barents Sea records 68 (NP05-11-70GC; 78.40°N, 32.42°E), as well as the re-emergence of IP 25 in JM09-KA11-GC.
In this study, we demonstrate that %C 37:4 is a reliable and sensitive indicator for paleo sea ice, and has the potential for quantitative sea ice reconstructions based on surface sediment calibrations. As alkenones are solely produced by Isochrysidales, %C 37:4 directly reflects proportion of Group 2i Isochrysidales within Isochrysidales community induced by sea ice, and is not affected by variations in total productivity or sedimentation rates as IP 25 . %C 37:4 is also less likely to be affected by diagenetic factors due to the strong diagenetic stability of alkenones, and does not suffer from dissolution like microfossil sea ice proxies such as diatoms, ostracods, and foraminifers. As an example of the durability of the %C 37:4 proxy, a gradual increase of %C 37:4 from 0 to 10% was observed in Bering Sea after the onset of MIS M2 glaciation (~3.3 Ma) in concordance with an increase in sea icerelated diatoms 50 . In addition, %C 37:4 provides site-specific sea ice information which is different from proxies such as methanesulfonic acid and sea salt in ice from glaciers and ice sheets that records the averaged sea ice condition at a variable (e.g., depending on wind direction and speed) sector of ocean. Future work combining molecular biology and organic geochemistry analyses in culture experiments and large-scale analyses of seawater, sea ice, and sediment samples will improve the %C 37:4 -sea ice calibration and shed light on other sea ice properties such as seasonality. NGS of Canadian Arctic Archipelago surface sediment samples. Sequencing of the surface sediment samples was performed by Jonah Ventrues, Boulder, CO, USA, (https://jonahventures.com/). Genomic DNA was extracted from the samples using the DNeasy PowerSoil HTP 96, then a two-step PCR protocol was performed. First, a portion of 18S rRNA was amplified by an adapted version of primer pair 528Flong and PRYM01+7 (F: GCGGTAATTCCAGCTCCAA, R: GATCAGTGAAAACATCCCTGG, Egge et al. 69 ). The first PCR step following the program described in Egge et al. 69 was performed in 25-μl reactions containing Phusion GC Buffer, 0.4 mM of each primer, 0.2 mM dNTP, DMSO 3%, 0.5 U Phusion polymerase (Thermo Scientific), and 2 µl of DNA template. After cleanup of the amplicons, a second round of PCR was performed to give each sample a unique 12-nucleotide index sequence. The final indexed amplicons were then cleaned and normalized using SequalPrep Normalization Kit (Life Technologies, Carlsbad, CA) and proceeded to sequencing on an Illumina MiSeq using V2 600-Cycle Kit (San Diego, CA). Sequencing success and read quality was verified using FastQC v0.11.8. The forward and reverse primers were removed from the sequences, and sequences with length below 100 bp were discarded. The ASVs were compiled for each sample after denoising by UNOISE3 algorithm with an alpha value of 5 to remove sequencing error 70 . Taxonomy of the ASVs was assigned using the SILVA reference database (including 506 Isochrysidales sequences) and assign_taxonomy.py as implemented in QIIME/1.9.1 71 .
Reanalyses of environmental sequences and phylogeny. We searched for shortread studies including the term "sea ice" on the European Nucleotide Archive, and runs from studies targeting subfragments of 18S rRNA that included sea ice or seawater samples were downloaded. Primers were removed from the downloaded raw reads using cutadapt 72 , except from Roche 454 sequenced reads where forward primers were removed after quality trimming. ASVs were compiled after the reads were quality trimmed using DADA2 73 , except for studies with available quality trimmed ASVs 42,74 . The DADA2 parameters were adjusted based on the quality profiles of the sequencing runs. Taxonomic affiliations of the ASVs were identified using the assignTaxonomy command in DADA2 and, as reference sequences, the Protist Ribosomal Reference database 75 (including 159 expert-curated Isochrysidales sequences from each taxonomic groups). Sequences classified to the order Isochrysidales were selected to proceed to further analysis. Close relatives of the environmental sequences were identified through BLAST search against NCBI GenBank and compiled as reference sequences for phylogeny. All sequences were then aligned against a curated alignment from Gran-Stadniczenko et al. 76 using PyNAST 77 . In total, the alignment contains 105 sequences, including 6 Coccolithales, 16 Groups 2 and 3 Isochrysidales cultures, 46 published environmental sequences from GenBank, and 37 recovered or reanalyzed environmental sequences from this study. A maximum-likelihood phylogeny was inferred using the RAxML 78 program with substitution model GTRCAT with 1000 bootstraps through CIPRES 79 .
Culture experiments. We obtained the RCC5486 strain (GenBank accession: MH764930) from the Roscoff Culture from the National Institute for Environmental Studies. The strain was acclimatized to 3 and 6°C for 10 days before the start of corresponding culture experiments. The f/2 medium was prepared from filtered and sterilized seawater collected from Vineyard Sound, Woods Hole, MA, USA, adjusted to 15,21,26,31, and 38 ppt. Cultures were grown under a light:dark cycle set at 16:8 h. The light intensity was 140 μE · m −2 · s −1 . Culture experiments under each salinity and temperature condition were triplicated.
Cultures were harvested at early stationary phase (monitored using hemocytometer counts (Hausser Scientific, PA, USA)) by filtering onto 0.7 μm glass fiber filters (Merck Millipore, MA, USA). All filters were wrapped with aluminum foils and immediately frozen at −20°C before further extraction and analysis. For alkenones analyses, all of the filters were freeze-dried overnight and then dissolved in dichloromethane (DCM) and sonicated (3 × 30 min, 40 ml each time). The total extracts were separated and analyzed for alkenones as described in the section "Alkenones analyses." Alkenones analyses. Surface sediment samples were analyzed for lipids by extracting around 10 g of freeze-dried sediment using a Dionex TM accelerated solvent extraction system with DCM:methanol mixture (9:1, v/v) to obtain a total lipid extract (TLE). The TLE was then separated into alkane, ketone, and polar fractions through silica gel columns by eluents with increasing polarity: hexane, DCM, and methanol. The ketone fraction containing alkenones was analyzed by gas chromatography-flame ionization detection Agilent 7890N Series instrument equipped with mid-polarity column RTX-200 (105 m × 250 μm × 0.25 μm) with the oven program described in Zheng et al. 80 . This method is able to fully eliminate the co-elution between alkenones and alkenoates, as well as co-elution between C 38 methyl and ethyl alkenones 80 . Alkenone peaks were identified and quantified based on retention time compared with standard Group 1 alkenone samples extracted from BrayaSø Lake (Greenland) through Agilent ChemStation B.03.02. A gas chromatography-mass spectrometer (GC-MS, Agilent 7890B interfaced to 5977 inert plus MSD) equipped with RTX-200 columns (105 m × 250 μm × 0.25 μm) was used to examine co-elution. Samples with compounds co-eluting with alkenones were purified using silver thiolate silica gel columns with hexane: DCM (1:1, v/v), DCM, and acetone 23 . Regressions of alkenone indices and environmental parameters were performed via MATLAB (R2018b).
Partially purified TOEs were resuspended in hexane and purified using column chromatography (SiO 2 ; ca. 0.5 g in a glass pipette), with fractions containing IP 25 collected using hexane (6 ml). Final purification was achieved using Ag-ion chromatography (Supelco Discovery ® Ag-Ion; 0.1 g). After elution of saturated hydrocarbons (hexane; 1 ml), unsaturated hydrocarbons including IP 25 were collected in acetone (2 ml). Identification and quantification of IP 25 in purified sediment extracts was carried out via GC-MS using established methods 81 with an Agilent 7890 gas chromatograph equipped with a HP 5MS fused-silica column (30 m; 0.25 µm film thickness; 0.25 mm internal diameter) coupled to an Agilent 5975 series mass spectrometric detector. IP 25 was identified by its characteristic retention index (RI HP5-MS = 2081) and mass spectrum. Quantification was carried out in single-ion monitoring mode. The resulting peak areas for IP 25 were normalized to internal standard responses, instrumental response factors, and sediment mass.
Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability
Raw sequence data generated in this study were archived in NCBI GenBank with accession number PRJNA650544, and assembled Isochrysidales sequences were archived in NCBI GenBank with accession number MT647637-MT647640. Reanalyzed NGS data can be found through European Nucleotide Archive with accession codes provided in Supplementary Data 2. Reference database SILVA is available at https://www.arb-silva.de/, and PR2 is available at https://github.com/pr2database/pr2database.
Received: 28 August 2020; Accepted: 12 November 2020; Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/.