Source identification of the Arctic sea ice proxy IP25

Abstract

Analysis of the organic geochemical biomarker IP₂₅ in marine sediments is an established method for carrying out palaeo sea ice reconstructions for the Arctic. Such reconstructions cover timescales from decades back to the early Pleistocene, and are critical for understanding past climate conditions on Earth and for informing climate prediction models. Key attributes of IP₂₅ include its strict association with Arctic sea ice together with its ubiquity and stability in underlying marine sediments; however, the sources of IP₂₅ have remained undetermined. Here we report the identification of IP₂₅ in three (or four) relatively minor (<5%) sea ice diatoms isolated from mixed assemblages collected from the Canadian Arctic. In contrast, IP₂₅ was absent in the dominant taxa. Chemical and taxonomical investigations suggest that the IP₂₅ -containing taxa represent the majority of producers and are distributed pan-Arctic, thus establishing the widespread applicability of the IP₂₅ proxy for palaeo Arctic sea ice reconstruction.

ARTICLE

Received 28 Feb 2014 | Accepted 22 May 2014 | Published 18 Jun 2014

DOI: 10.1038/ncomms5197

Source identication of the Arctic sea ice proxy IP25

T.A. Brown1, S.T. Belt1, A. Tatarek2 & C.J. Mundy3

Analysis of the organic geochemical biomarker IP25 in marine sediments is an established method for carrying out palaeo sea ice reconstructions for the Arctic. Such reconstructions cover timescales from decades back to the early Pleistocene, and are critical for understanding past climate conditions on Earth and for informing climate prediction models. Key attributes of IP25 include its strict association with Arctic sea ice together with its ubiquity and stability in underlying marine sediments; however, the sources of IP25 have

remained undetermined. Here we report the identication of IP25 in three (or four) relatively minor (o5%) sea ice diatoms isolated from mixed assemblages collected from the Canadian

Arctic. In contrast, IP25 was absent in the dominant taxa. Chemical and taxonomical investigations suggest that the IP25-containing taxa represent the majority of producers and are distributed pan-Arctic, thus establishing the widespread applicability of the IP25 proxy for palaeo Arctic sea ice reconstruction.

1 Biogeochemistry Research Centre, School of Geography, Earth and Environmental Sciences, University of Plymouth, Plymouth PL4 8AA, UK. 2 Institute of Oceanology Polish Academy of Sciences, Powstacw Warszawy 55, 81-712 Sopot, Poland. 3 Centre for Earth Observation Science, University of Manitoba, 535 Wallace Building, 125 Dysart Road, Winnipeg, Canada R3T 2N2. Correspondence and requests for materials should be addressed to T.A.B.(email: mailto:[email protected]

Web End [email protected] ).

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The reconstruction of sea ice conditions in the polar regions represents a key objective within palaeoceanography and palaeoclimatology1. This is largely owing to the major role

that sea ice has in controlling the energy budget at the Earths surface, due to its high albedo, and also since it limits heat, gas and moisture exchange between the oceans and the atmosphere2,3. Further, sea ice contributes to ocean ventilation and circulation via brine rejection and freshwater input, following formation and melting, respectively2.

Performing such reconstructions is challenging, however, since sea ice itself leaves no direct legacy signature in geological archives, necessitating the use of so-called proxy methods. A number of proxies for sea ice exist and these are both biogenic1 and non-biogenic in origin46. Within the former category, the recent discovery of the organic geochemical biomarker IP25

(ref. 7) (a C25 highly branched isoprenoid (HBI) lipid8) has attracted considerable interest, not least because it possesses the unique attribute of being produced within the sea ice itself. Consistent with this sea ice origin, the occurrence of IP25 in

marine sediments shows a strong correlation, spatially, to seasonally ice-covered Arctic waters9, and, from a temporal perspective, IP25 appears to be stable in sediments for millions of years10. Combined, these attributes of IP25 have provided the foundation for decadal to millennial-scale sea ice reconstructions across the Arctic915.

Despite these interesting and valuable applications of the IP25

sea ice proxy, the source organisms responsible for formation of this lipid in sea ice have remained elusive. A diatom source has been proposed7,16, however, since IP25 has been reported in

Arctic sea ice biota dominated by diatoms16,17 and similar lipids occur in some non-sea ice algae1822. More specically, it has been hypothesized that sea ice diatoms belonging to the Haslea genus are likely producers of IP25 on the basis of biosynthesis of related biomarkers by such species7,16,17,2325. Nevertheless, IP25 has never been reported in any cultures of diatoms, including some Haslea species isolated from Arctic sea ice9. Arguably, until the sources of IP25 have been determined, together with information regarding their distributions across the Arctic, the potential for IP25 as a palaeo sea ice proxy cannot be fully realized.

In the current study, we identify three (or four) sea ice endemic diatom species that are responsible for IP25 production in

Canadian Arctic sea ice during a spring bloom. We achieve this by isolation of individual cells from mixed sea ice diatom assemblages and analysis of their lipid composition by gas chromatographymass spectrometry (GCMS). The identied IP25 producers (Pleurosigma stuxbergii var. rhomboides (Cleve in

Cleve and Grunow) Cleve, Haslea kjellmanii (Cleve) Simonsen,H. crucigeroides (Hustedt) Simonsen and/or H. spicula (Hickie) Lange-Bertalot) constitute a relatively minor proportion of the ice taxa, but, signicantly, they are nonetheless common pan-Arctic species that validate the notion that IP25 represents a widespread proxy for palaeo Arctic sea ice.

ResultsIdentication of IP25 producers. Taxonomic assessment of the diatom assemblage within several algal aggregates from Resolute Bay in the Canadian Arctic revealed a distinctive ora, characteristic of sea ice2628. Thus, the three most abundant species were Navicula pelagica (ca. 4050%), Nitzschia frigida (ca. 1020%) and Pauliella taeniata (ca. 1020%) (Table 1), with a near absence of planktic cells (o0.5%). From one assemblage (SIA-1), we isolated and combined sufcient numbers of cells from (at least) six individual species to perform quantitative lipid analysis by GCMS to show that IP25 was present in P. stuxbergii

var. rhomboides and at least two species from the genus Haslea (H. kjellmanii, H. crucigeroides and/or H. spicula) (Fig. 1), but was absent in other important ice algal species; N. pelagica,N. frigida, and Entomoneis paludosa (Fig. 1 and Table 2).

The occurrence of IP25 in H. crucigeroides (and/or H. spicula)

and H. kjellmanii is consistent with the production of other HBIs by members of the genus Haslea18,19,25,2931, which led to the previous suggestion that Haslea was a likely source of IP25 in

Arctic sea ice7,16,23,24, despite the failure for such species to produce IP25 in culture29. The identication of P. stuxbergii var.

rhomboides as an IP25 producer is also consistent with the formation of other HBIs by several Pleurosigma diatoms20,32, although we are unaware of any reports describing the HBI content of P. stuxbergii var. rhomboides, per se. In contrast, IP25

was absent in at least two of the typically abundant ice ora (viz,N. pelagica and N. frigida), supporting the notion that IP25 is

produced selectively by a limited number of Arctic sea ice diatom taxa7,16,23,24. Indeed, the same conclusion was reached following taxonomic analysis and lipid characterization of mixed diatom assemblages in sectioned sea ice cores collected from the same location as the samples described here16.

Previously, it has been shown that, although the physiology of diatom genera can be important for production of HBIs, not all species within such genera are HBI producers. Thus, some species within the Haslea, Pleurosigma and Navicula genera are known HBI producers, but others are not. For example, species such as H. ostrearia18 and P. intermedium32 produce HBIs, butH. wawrikae and P. angulatum do not29. Within the current context, we note that IP25 was identied in H. kjellmanii, but was absent in H. vitrea (Table 2). IP25 was also found in H. crucigeroides and/or H. spicula; however, since H. crucigeroides and/or H. spicula could not be distinguished during the cell isolation (see Methods section), we are unable to conclude whether both (or only one) of these are IP25 producers. IP25 and other HBIs were absent in cells of N. pelagica (Table 2), despite the production of HBIs by some species of Navicula22. The absence of IP25 in N. frigida is not surprising, however, since there have been no reports of HBI production in the genus Nitzschia.

In addition to IP25, the structurally related di-unsaturated HBI biomarker C25:29 was also identied in each of the IP25-producing species (Table 2). Previous studies based on the analysis of IP25 in

sea ice and sediments have shown a consistent abundance relationship between these two structural homologues indicating a common source at least within the Arctic12,24,33,34. Our data not only conrm this source association but, the similarity of the C25:2/IP25 ratio in producers (2.30.8) to that found in sea ice and sediments12,24,33,34, implies a close link between the source and Arctic sedimentary signatures of these two biomarkers. As such, a signicant formation of IP25 over C25:2 (or vice versa) in sea ice or differential degradation of either biomarker in situ, seems unlikely. Finally, the ranges of intracellular concentrations of IP25 and C25:2 in the isolated sea ice diatoms were similar between individual species (Table 2) and to those of HBIs measured in culture16.

Major IP25-producing taxa. The species selected for lipid analysis corresponded to B65% of the total taxa in SIA-1, with IP25 producers representing only 1.8% of the total. As such, the absence of IP25 in the abundant N. pelagica and N. frigida corresponded to B63% of total cell numbers. Although not all species were investigated (mainly because of low cell numbers of many minor species), these data suggest that the majority of sea ice diatom cells do not synthesize IP25 (Tables 2 and 3), which is consistent with previous lipid-based estimates that indicated only the minority of sea ice ora (15%) likely contribute to IP25

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Table 1 | Taxonomic composition of diatom aggregates.

SIA-1 SIA-2 SIA-3 SIA-4 Amphora laevissima 1.7 1.9 2.0 0.9

Attheya septentrionalis 0.7 1.3 0.4 0.9 Bacillaria paxillifera 1.1 0.5 Coscinodiscus centralis 0.1 Cylindrotheca closterium 0.1 0.2 Entomoneis gigantean 0.4 0.2 Entomoneis kjellmanii 0.4 0.4 0.4 1.0 *Entomoneis paludosa 2.0 2.0 3.8 2.2 Fragilariopsis cylindrus 3.9 8.4 7.2 1.3 Gyrosigma tenuissimum 0.7 *Haslea crucigeroides (and/or Hasleaspicula)

0.4 0.5

*Haslea kjellmanii 0.4 0.7Haslea kjellmanii (empty frustule) 0.1 0.2 *Haslea vitrea 0.1Melosira sp. 0.2 Navicula algida 0.2 0.1 0.1 Navicula cf. trigonocephala 0.1 Navicula directa 3.0 3.3 0.4 2.4 Navicula kariana 0.6 0.1 0.2 0.3 Navicula obtuse 0.2 0.4 0.7 0.1 *Navicula pelagica 39.0 44.8 52.8 Navicula septentrionalis 0.4 0.4 0.2 Navicula superba 0.2 Navicula transitans 0.6 0.8 2.7 0.6 Navicula trigonocephala 0.2 0.7 Nitzschia arctica 3.1 2.5 6.7 Nitzschia brebissonii var borealis 0.1 *Nitzschia frigida 22.1 9.5 14.3 8.7 Nitzschia laevissima 0.4 1.1 4.5 0.9 Nitzschia neofrigida 0.7 4.2 2.3 Nitzschia pelagica 14.3 Nitzschia promare 0.1 1.1 Nitzschia seriata 0.1Nitzschia sp. 0.4 Pauliella taeniata 13.0 11.5 18.0 16.1 Phaeocystis sp. 0.6 Pinnularia quadratarea 1.5 1.1 4.0 2.0 Pinnularia semiinata 0.2 0.1Plagiotropis sp. 0.1 Pleurosigma sp. 0.1 *Pleurosigma stuxbergii var. rhomboides 0.4 0.8 0.2 0.1 Pseudogomphonema arcticum 3.0 1.7 4.0 2.3 Pseudogomphonema groenlandicum 1.1 0.2 2.7 1.0 Stenoneis inconspicua 0.4 0.4 Synedropsis hyperborean 0.6 2.1 1.8 0.5 Thalassiosira sp. 0.2 0.1 Other microorganisms 0.2 0.2 0.3

Total (%) IP25-producing cells 1.8 3.6 2.7 0.3

Abundances (%) of diatoms identied in ice algal aggregates. IP -producing species are indicated in bold.*Species analyzed for IP .

IP25

Haslea crucigeroides (and/or Haslea spicula) (empty frustule)

IP25 laboratory standard

Haslea crucigeroides/spicula

Pleurosigma stuxbergii var. rhomboides

Navicula pelagica

Nitzschia frigida

Entomoneis paludosa

Retention time

Figure 1 | Lipid extracts from isolated diatoms. Structure of IP25 and

partial GCMS (selective ion monitoring; m/z 350.3) chromatograms of lipid extracts obtained from various diatoms isolated from the mixed assemblages together with that of an authentic sample of IP25 (ref. 57).

1.4 2.4 1.8 0.2

biosynthesis7,16. Of course, it remains feasible that some other minor taxa may also be IP25 producers but, given the low percentages of all unexamined species, together with the consistency in cellular abundances of those taxa that are producers, we suggest that their contributions are not signicant. In support of this, we note that none of the unexamined genera (Table 1) are known HBI producers.

In order to conrm that H. crucigeroides (and/or H. spicula),H. kjellmanii and P. stuxbergii var. rhomboides indeed represented the majority (at least) of the IP25-producing species, and

that the unanalyzed cells were not signicant contributors to the IP25 budget, we calculated the HBI abundance of SIA-1 relative to the total organic carbon (TOC) content (HBI/TOC 0.05%) and

compared this ratio with the corresponding value obtained from a previously known HBI-producing species (H. ostrearia) in culture (HBI/TOC 1.4%). The B30 times lower contribution of HBIs

to sea ice algal TOC compared with that found for cultured diatoms provides a reasonable estimate of the percentage of HBI-producing diatoms in the sea ice algal assemblage (B3.6%).

This low percentage conrms the relatively small proportion of IP25 producers in the total assemblage, while the close similarity of this estimate to the combined percentages of H. crucigeroides (and/or H. spicula), H. kjellmanii and P. stuxbergii var. rhomboides between SIA-14 (Table 2) indicates that these species represent the majority (if not all) of the IP25 producers.

DiscussionHaving identied the major contributors of IP25 in sea ice from a

single study location in the Canadian Arctic, we next aimed to address the signicance of this discovery with respect to the wider scale applicability of the IP25 biomarker for palaeo sea ice studies.

To do this, we compiled literature accounts of P. stuxbergii var. rhomboides, H. kjellmanii, H. crucigeroides and H. spicula (note: the latter two as separate species), and compared ndings with reports of IP25 in sediments (and sea ice). A summary of the spatial relationship between the two is shown in Fig. 2.

According to Poulin35, Haslea and Pleurosigma are typical of sea ice biota with ve common taxa between them.H. crucigeroides, H. kjellmanii and H. spicula are identied as Arctic/sub-Arctic in distribution35, while H. crucigeroides andP. stuxbergii var. rhomboides are the most commonly reported (Table 1). Owing to the close similarity betweenH. crucigeroides and H. spicula36, their occurrences may have been combined in some investigations, as has been the case here for the cell isolation and extraction experiments (see Methods section). Despite their widespread occurrence, however,H. crucigeroides, H. kjellmanii, H. spicula and P. stuxbergii var. rhomboides are always relatively minor taxa, with abundances usually o5% (Table 3), as we found here (Table 1). Individual studies have reported these IP25-producing species in rst-year

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Table 2 | HBI content of isolated cells.

Diatom species Contribution to assemblage (%) No. of cells isolated IP25 (pg cell 1) C25:2 (pg cell 1) Haslea crucigeroides (and/or Haslea spicula) 1.4 200264 0.63.4 1.510.3 Haslea kjellmanii ND 4049 1.93.3 4.79.0 Haslea vitrea ND 25 Pleurosigma stuxbergii var. rhomboides 0.4 100120 0.63.8 0.610.7 Entomoneis paludosa 2.0 201 Navicula pelagica 39.0 200 Nitzschia frigida 22.1 220

indicates not detected. ND, not determined.

Table 3 | Reported presence of IP25-producing diatoms in sea ice and IP25 in Arctic sediments.

Study region Year of study Occurrence of IP25-producing diatoms Literature source1 2 3 4 5 Combined abundance (%) Species identication IP25

Cape Eglinton 1890s |* |* |* |* P 58 23 West Greenland 1890s |* |* |* |* P 58 44

Canada Basin 1893 |* |* |* |* P 58 Makarov Basin 1894 |* |* |* |* P 58

Barrow 1964 |* |* P 59 23 Davis Strait 1978 | | P 37 23

Beaufort Sea 1980 |* |* 15 60 61 Manitounuk Sound B1980 | | P 62 63

Barents Sea 1985 | 15 28 42,43 Resolute Bay 1989 | | | o1 28 7,40

Laptev Sea 198790 | | | P 38 24,34 East Siberian Sea 198790 | | | P 38 23

Chukchi Sea 198790 | P 38 23 N/E Svalbard 1991 | o1 28 64

Franz Josef Land 1991 | | | P 39 24 NE Water Polynya 1993 | | | 15 65 44

West Greenland 1995 | o1 28 White Sea 200103 | | | P 66

McDougal Sound 2001 | | | 0.6 67 7,16 McDougal Sound 2003 | | | | 2.6 7 7,16

Franklin Bay 2004 | | 0.9 68 69 Central Arctic 2004 | | 13 49

Churchill 2005 | 2.5 7 7 Resolute Bay 2011 | | | | 6.1 70 16

Mean 2.2

No published data from this region; P, present but not quantied.

Occurrences of H. crucigeroides (1), H. spicula (2), H. kjellmanii (3), H. spp (4) and P. stuxbergii var. rhomboides (5); and combined percentages identied in taxonomic studies of Arctic sea ice. The approximate study locations are indicated in Fig. 2. Reports of occurrences of IP from nearby locations are also indicated.*Historical synonym.

sea ice for locations ranging from 58 to 87oN, including coastal, shelf and deep ocean environments. Further, such species have been reported in sea ice of varying thickness (for example, 4300 cm)37,38 and type28,39.

With respect to biomarker-based studies, IP25 has also been reported in surface and downcore sediments from across the Arctic, as reviewed by Belt and Mller9, while here, we demonstrate the excellent spatial relationship between occurrences of IP25 in Arctic surface sediments (and sea ice)

and reports of the IP25-producing diatoms identied in the current study (Table 3 and Fig. 2). For example, H. crucigeroides,H. kjellmanii, H. spicula and P. stuxbergii var. rhomboides have been identied in sea ice from across the Canadian Arctic and sub-Arctic (Table 1), and IP25 has been identied as a common component in surface sediments from these regions, with downcore abundances providing the basis for Holocene sea ice reconstructions9,13,40,41. Similarly, in the eastern Arctic, whereH. crucigeroides, H. kjellmanii, H. spicula and P. stuxbergii

var. rhomboides are also common, IP25 occurrence in recent sediments from the Kara, Laptev and Barents Sea shows a strong correlation with modern sea ice cover24,42, and this has aided the reconstruction of Holocene (and older) sea ice records from these regions10,43. Further, the observation of IP25-producing diatoms in sea ice from around Svalbard and the North East Polynya (NE Greenland) is consistent with the occurrence of IP25 in nearby

surface sediments42,44, and the longest IP25-based palaeo sea ice records to date are also from this region10,45. The majority of these IP25 studies have been conducted in relatively shallow marine settings, proximal to continental shelves while, in contrast, there is currently a paucity of data from deeper oceanographic settings such as the Greenland Sea or the central Arctic Ocean. For the latter, in particular, it has been suggested previously that the combined presence of thick multi-year ice, likely unsuitable for diatom growth, and low sediment accumulation rates may somewhat limit the application of the IP25 proxy method for palaeo sea ice reconstruction9. However,

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0

60N

70N

80N

Greenland

90E

90W

Russia

Canada

180E

Figure 2 | Distribution of IP25 producers and presence of IP25 in sediments. Summary map showing locations where IP25 has been identied in Arctic surface sediments (yellow circles) and sea ice (orange circles) (Supplementary Data 1). Red circles indicate the approximate locations of sea ice taxonomic studies reporting Haslea spp. and Pleurosigma stuxbergii var. rhomboides (summarized in Table 1). The pink square shows the collection location of the ice algal aggregates analyzed in the current study. The black line shows the median March sea ice extent (National Snow and Ice Data Center; 19812010).

Figure 3 | IP25-producing diatom species. Scanning electron micrographs of IP25-producing diatoms identied in SIA-1. (a) Pleurosigma stuxbergii var. rhomboides; (b) Haslea crucigeroides (and/or Haslea spicula);

and (c) Haslea kjellmanii. Scale bars, 10 mm.

within the context of recent climate change and a reduction in both Arctic sea ice extent and thickness46,47, in particular, multi-year ice is becoming increasingly replaced by thinner rst-year ice48. Consistent with such a change, we note that some of the IP25 producers identied here have, in fact, been reported in rst-year ice within the central Arctic (87oN)49, and IP25 has recently been detected in surface sediments 480oN (X. Xiao

and R. Stein, personal communication). We suggest that such observations, in combination with our results here, will likely expand the potential for the IP25 proxy to provide palaeo sea ice reconstruction data for the entire Arctic.

Previously, the identication of IP25 in Arctic sediments has been interpreted as proxy evidence for past seasonal sea ice cover, with variations in sedimentary abundance attributed to corresponding changes in sea ice; an approach supported by a number of meaningful reconstructions9. In contrast, the possible inuence of ecological controls over IP25 production and their potential impact on its sedimentary abundance have been largely ignored, although its likely importance has been alluded to7,9,16. Having now identied those species that are responsible for IP25

formation, it should be possible, in the future, to not only discuss ecological factors when interpreting sedimentary IP25

distributions, but also to test the signicance of these

experimentally, and in an informed manner.

Here, we suggest that the relatively consistent contribution of IP25 producers to mixed Arctic sea ice diatom assemblages (Table 3) provides some evidence that the larger temporal changes in IP25 concentration often seen in sediments9,12,13,40,45 are more likely attributable to sea ice variations than major modications to diatom assemblage composition, although changes in overall production may still be important. On the other hand, relatively small or subtle variations in sedimentary abundance may, potentially, simply reect minor changes in species composition or overall production rather than variations in sea ice cover. What is now clear, however, is that since IP25

production is species specic and restricted to the minority

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diatom taxa, conclusions from future investigations into environmental or phenotypic variables over IP25 production will require investigations of specic species, probably via in situ measurements in the eld, rather than whole assemblage or simple biomass determinations. In this respect, the discovery of the IP25 producers represents a key step in determining the factors that control the production and fate of IP25, which have been identied as important for its development as a palaeo sea ice proxy9.

Methods

Sample collection. Four oating ice algal aggregates (SIA-14) were collected from a sampling hole cut in rst-year ice at Resolute Bay in association with the Arctic-ICE (Ice Covered Ecosystems) project (19 June 2012; 74o43.6130N;

95o33.4960W; Fig. 2). Aggregates consisted of sea ice algal assemblages recently sloughed from the underside interstitial channels of rst-year ice and were stored in Whirl-Pak bags and frozen ( 20 C). A taxonomic description of the content of

sectioned ice cores collected from the same location is given elsewhere16.

Species identication. Taxonomic identication of diatom species was carried out on each aggregate (Table 1). Sub-samples of each of SIA-14 were freeze-dried, and B10 mg of dried material was re-suspended in 100 ml of articial seawater.

Aliquots (0.5 ml) were taken for cell enumeration using the Utermhl method50. Cell counts (400600) were performed on parallel transects using an inverted microscope (Nikon Ti-S) at 60 magnication51,52.

More detailed examination of certain taxa was achieved by dry-mounting sub-samples of cleaned (10% HCl; 70 C for 30 min and 3 10 ml Milli-Q washes) cells

and examination using a JEOL 7001 F scanning electron microscope. Specically, diatoms belonging to the genus Haslea (Fig. 3) were identied based upon general morphological dimensions in addition to features considered characteristic of the genus including, for example, the presence of external longitudinal strips over many areolae, with intervening continuous slits35,53,54. Additional characteristic features included elongated helictoglossae, a well-dened accessory rib on the primary side of the raphe sternum and typically straight external raphe ssures with only slight terminal deection/expansion53. Although both H. crucigeroides and H. spicula were identied in the assemblages as part of the detailed taxonomic analysis (Tatarek, Poland), it was not possible to distinguish between these two species during the low magnication ( 40) cell isolation stage (Brown, UK)

because of the close similarities between them36. For P. stuxbergii var. rhomboides, identication was conrmed from the characteristic sigmoidal valve displaying ne striae, with slightly denser oblique than transverse pattern55 and equally thickened primary and secondary raphe sternum (cf. Gyrosigma54; Fig. 3).

Total organic carbon. Sub-samples (B50 mg) of freeze-dried algae were decarbonated (10% HCl; 10 ml), washed (3 10 ml Milli-Q water) and freeze-dried

( 80C; 0.001 mbar; 24 h) before analysis using a Thermoquest EA1110 CHN

analyser. L-cystine was used as a calibration standard.

Extraction and analysis of lipids. Lipids were extracted from bulk algal aggregates and combined cells of individual species. For analysis of bulk aggregates, B50 mg of each aggregate was rst washed (3 10 ml Milli-Q water) to remove marine salts

before being freeze-dried ( 80 C; 0.001 mbar; 24 h) and re-weighed before

extraction. For extractions of individual diatom species, non-washed aggregate sub-samples were re-suspended in B3 ml ltered (0.2 mm) articial seawater (deionized water; 32 p.p.t. Tropic Marin salt) in a clean glass Petri dish. Individual diatom cells were identied using a Nikon TS2000 inverted light microscope( 10 and 40 objectives) in phase contrast and isolated manually using a

modied Pasteur pipette.

Following addition of an internal standard (9-octylheptadec-8-ene; 2 mg), bulk aggregates were saponied (20% KOH; 80 C; 60 min) and extracted with hexane according to Brown et al.17, whereas for isolated cells a total hexane extract only was obtained (hexane; 3 1 ml, ultrasonication; 3 5 min). In each case, the

resulting total hexane extract suspensions were ltered through pre-extracted (dichloromethane/methanol) cotton wool to remove cells before being partially dried (N2 stream) and fractionated into non-polar lipids by column chromatography (hexane (3 ml)/SiO2).

Analysis of partially puried non-polar lipids was carried out using GCMS techniques56 with minor modications to increase instrument sensitivity. Identication of IP25 was achieved by a characteristic mass spectral response (m/z 350.3; Fig. 1) using selective ion monitoring and co-injection of extracts with an authentic standard of IP25 (ref. 57). Quantication was achieved by integrating the m/z 350.3 ion responses of IP25 and the internal standard in selective ion monitoring mode, and normalizing the ratio between them using an instrumental response factor56 and the number of cells extracted.

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Acknowledgements

We thank the University of Plymouth for a Fellowship to T.A.B. We also thank J. Wiktor (Institute of Oceanology, Polish Academy of Sciences) and Michel Poulin (Canadian Museum of Nature) for their taxonomical assistance. We also thank Peter Bond at the University of Plymouth Electron Microscopy Centre for assistance with the scanning electron microscope. We are especially grateful to Michel Gosselin (Universit du Qubec Rimouski) for providing additional eld support under the Arctic-ICE programme, and Alexis Burt (University of Manitoba) for collecting aggregate samples. Sample collection was supported through a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery grant to C.J.M., and by the Polar Continental Shelf Program (PCSP) of Natural Resources Canada. This is a contribution to the research programs of ArcticNet, Arctic Science Partnership (ASP) and the Canada Excellence Research Chair unit at the Centre for Earth Observation Science.

Author contributions

T.A.B. designed and implemented the study, performed lipid analyses and data synthesis; A.T. carried out taxonomic assessments of samples; T.A.B. and S.T.B. wrote the manuscript with signicant input from C.J.M.

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How to cite this article: Brown, T. A. et al. Source identication of the Arctic sea ice proxy IP25. Nat. Commun. 5:4197 doi: 10.1038/ncomms5197 (2014).

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