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Characterization of unusual sterols and long chain diols, triols, keto-ols and n-alkenols in El Junco Lake, Galápagos Article in Organic Geochemistry · January 2013 DOI: 10.1016/j.orggeochem.2013.11.004
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Organic Geochemistry 66 (2014) 80–89
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Characterization of unusual sterols and long chain diols, triols, keto-ols and n-alkenols in El Junco Lake, Galápagos Alyssa R. Atwood a,⇑, John K. Volkman b, Julian P. Sachs a a b
University of Washington, School of Oceanography, Seattle, WA 98195, USA CSIRO Marine and Atmospheric Research, GPO Box 1538, Hobart, Tasmania 7001, Australia
a r t i c l e
i n f o
Article history: Received 16 May 2013 Received in revised form 25 October 2013 Accepted 5 November 2013 Available online 15 November 2013
a b s t r a c t A variety of lipid biomarkers were identified in sediments from El Junco Lake, Galápagos and their sources investigated for potential use in paleoclimate applications. A series of unusual sterols was also found, including 4a-methylgorgostanol, reported in only four species of dinoflagellates to date. We also tentatively assigned 22,23-methylene-4a-methyl-24-ethylcholest-5-en-3b-ol, the mass spectrum of which matched a sterol found in resting cysts of the dinoflagellate Peridinium umbonatum. In addition, we identified the novel sterol 4a,22,23,24-tetramethyl-5a-cholest-22E-en-3b-ol. Based on the unique sterol distribution, we hypothesize that a dinoflagellate from the genus Peridinium was the primary source of dinosterol and the novel sterols throughout the sediment record. The source specificity and abundance throughout the 3.7 m of recovered sediment make dinosterol an excellent target for hydrogen isotope analysis for use as a paleohydrological proxy in future studies. The abundant C30 and C32 1,x20-diols and keto-ols, C29 9,10-diol and C29 1,x9,x10-triol likely derive from the ferns Azolla microphylla and Cyathea weatherbyana, while sources of the C30 1,x16-diol and keto-ol, C32 1,x18-diol and keto-ol, and the C30–C32 n-alken-1-ols are likely limited to aquatic microalgae. Due to their source specificity, these diol, triol, keto-ol, and n-alkenol biomarkers present further tools for studying past environmental and climatic change. ! 2013 Elsevier Ltd. All rights reserved.
1. Introduction Lake sediments are an important tool with which to study past environmental and climatic change. Changes in the chemical, biological and physical characteristics of lacustrine sediments provide insight into changes in the biological community, surrounding vegetation and hydrologic flux in lakes, all of which can be used to infer past changes in local climate. Lake sediments are a particularly important paleoclimate tool in many areas of the tropics, where other common paleoclimate proxies, including tree rings and ice, are limited. El Junco Lake lies atop a caldera in the highlands of San Cristóbal Island, Galápagos (1"S, 89"W) at 670 m elevation. It is the only permanent freshwater lake in the Galápagos Islands, with a diameter of 280 m and maximum depth of 6 m. It is fed by heavy rain during the rainy season and during El Niño events, and by fog associated with the persistent stratus clouds blanketing the highlands. It has conductivity 20–27 lS/cm, temperature 14–24 "C, pH ca. 5.5 and is mesotrophic, as given by total phosphorus of 18 TP/l and a Secchi depth of 2.5–3.6 m (Colinvaux, 1968; Ferrington and Pehofer, 1996). Due to the presence of strong easterly trade winds and
⇑ Corresponding author. Tel.: +1 206 694 3143; fax: +1 206 685 3351. E-mail address:
[email protected] (A.R. Atwood).
0146-6380/$ - see front matter ! 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.orggeochem.2013.11.004
the relatively shallow depth, the lake is well mixed, as indicated by uniform temperature and O2 profiles. The lake is ideally suited for studies of past climate change as it is located in a region of the tropical Pacific where key ocean–atmosphere interaction occurs and which influences climate on a global scale. Sediment from the lake, dating back to ca. 10 kyr BP, has been used for paleoclimate analysis based on the physical characteristics of the sediment (Colinvaux, 1972; Conroy et al., 2008), assemblage of pollen and diatoms (Colinvaux, 1972; Colinvaux and Schofield, 1976a; Conroy et al., 2008) and hydrogen isotope composition of algal lipids (Sachs et al., 2009). The distribution and hydrogen isotope values of lipid biomarkers from lake sediments are an increasingly important tool for the study of past climate change (Sachs et al., 2009; Smittenberg et al., 2011; Sachse et al., 2012), but require knowledge of the biological sources of the biomarkers. For instance, it is known that large differences in hydrogen isotope fractionation can occur during lipid biosynthesis between different groups of aquatic algae (Zhang and Sachs, 2007). Because of this, source-specific biomarkers are highly valuable targets for paleoclimate applications. Unfortunately, the biological community of the lake has not been well studied and thus information on likely sources of the sedimentary biomarkers is limited. A variety of lipid biomarkers in the sediment of El Junco Lake have been characterized by Zhang et al. (2007, 2011, 2013). The Zhang et al. (2011) study found a variety of long chain diols, triols,
A.R. Atwood et al. / Organic Geochemistry 66 (2014) 80–89
keto-ols and n-alkenols as well as dinosterol, a biomarker widely used for paleoclimate studies (e.g. Ohkouchi et al., 1997; Sauer et al., 2001; Makou et al., 2010; Castañeda et al., 2011; Smittenberg et al., 2011) due to its ubiquitous nature and the fact that it is produced almost exclusively by dinoflagellates. In order to evaluate the suitability of these biomarkers for paleoclimate applications, we have identified here the sources of dinosterol and the long chain diols, triols, keto-ols and n-alkenols by analyzing the lipid composition of both the sediments and surrounding vegetation. In addition, we have identified a variety of unusual sedimentary sterols and investigate their sources. 2. Methods 2.1. Sediment collection Cores were collected in September 2004 from 6 m water depth. An adapted Livingston-type corer (7 cm diameter) recovered the undisturbed sediment–water interface down to 64 cm, while a Nesje piston corer (9 cm diameter) allowed recovery of deeper sediment in ca. 100 cm intervals to 372 cm. Unconsolidated interface material was sectioned at 1 cm intervals on site and was stored frozen prior to analysis. The remainder of the sediment was transported as full cores to the lab where they were split, imaged, placed on a common depth scale and subsampled in 0.5 or 1 cm increments at 5 cm intervals. 2.2. Sediment dating A depth-age model was constructed from 210Pb and 14C measurements, a constant rate of supply model and linear interpolation between data points. Based on themodel, the 3.7 m sediment record spanned from present to 9.1 kyr BP. 2.3. Vegetation samples Twelve aquatic and terrestrial plants were identified by P. Colinvaux (see below) as the principle vegetation species in the El Junco Lake catchment and were sampled during his 1966 and 1972 field campaigns. Terrestrial plants included Cyathea weatherbyana (tree fern), Miconia robinsoniana (2–3 m flowering shrub), Cuphea carthagenensis (flowering herb), Ludwigia leptocarpa (flowering shrub), Polygonum punctatum (flowering herb) and Psidium galapageium (guava tree). Aquatic and semi-aquatic plants included Azolla microphylla (water fern from the shallows around the lake edge), Utricularia foliosa (submerged carnivorous water plant), Eleocharis mutata (emergent sedge bordering the lake), Hydrocotyle galapagensis (semi-aquatic herb around the lake edge and in the pasture outside the crater) and Najas guadelupensis (submerged water plant in streams below the crater; Colinvaux and Schofield, 1976a,b; Ricke, 2004). Air-dried plant samples were divided into two sets, one retained by P. Colinvaux in his private herbarium and the other sent to I. Wiggens at the Dudley Herbarium at Stanford University for identification (personal communication). Subsamples of P. Colinvaux’s private collection were used for lipid analysis. More information on the collection and preparation of vegetation samples is given by Ricke (2004). 2.4. Lipid biomarker extraction and initial purification Vegetation samples were first rinsed with dichloromethane (DCM) to remove surface contaminants, crushed, packed with sand and extracted using a soxhlet (DCM/MeOH, 9:1 v/v, 15 h). A known amount of 5a-cholestane was added to the extracts and highly
81
polar compounds were removed by applying the extract to a pipet column filled with 5% silica deactivated with EtOAc. The solvent was removed from the neutral fraction under a gentle N2 stream and the residue derivatized with 20 ll pyridine/20 ll bis(trimethylsilyl)trifluoroacetamide (BSTFA) at 60 "C for 20 min. The derivatized samples were diluted in EtOAc prior to gas chromatography–mass spectrometry (GC–MS). Sediment samples were freeze-dried and heneicosanol (n-C21 added as a recovery standard prior to accelerated solvent extraction (Dionex ASE 200) using three cycles of DCM/MeOH (9:1 v/v) at 100 "C and 1500 psi. The solvent was removed from the total lipid extract (TLE) under a gentle N2 stream using a Turbo-vap system (Caliper, Hopkinton, MA, USA). Neutral and polar lipids were separated from sediment extracts using aminopropyl SPE cartridges (Burdick & Jackson, 500 mg/ 4 ml) with DCM/isopropanol (3:1 v/v), followed by 4% HOAc in Et2O. The neutral fraction was applied to a column of 5% waterdeactivated silica gel and separated into hydrocarbon, wax ester, sterol/alcohol and polar fractions with hexane, 10% EtOAc in hexane, DCM and MeOH, respectively. 2.5. Lipid biomarker purification For further purification of lipid biomarkers, the sterol/alcohol fraction of sediment samples was subjected to normal phase (NP)- or reverse phase (RP)-high performance liquid chromatography (HPLC). An Agilent 1100 HPLC instrument with an integrated autoinjector, quaternary pump and fraction collector, coupled to an Agilent 1100 LC/MSD SL mass spectrometer with a multimode source operated in positive atmospheric pressure chemical ionization (APCI+) mode was used. A HPLC pump (Waters 510) delivered additional solvent (isooctane for NP-HPLC and MeOH for RP-HPLC) at 0.3 ml/min to the mass spectrometer for optimal ionization efficiency. For NP-HPLC, the sterol/alcohol fraction was dissolved in 100 ll of 15% DCM in hexane for injection. Dinosterol was eluted from a Prevail Cyano column (250 mm ! 4.6 mm ! 5 lm) with a mobile phase of 15% DCM in hexane at 1.5 ml/min. During RP-HPLC, the sterol/alcohol fraction was dissolved in 25 ll DCM/MeOH (2:1 v/v) and injected onto an Agilent ZORBAX Eclipse XDB-C18 column (250 mm ! 4.6 mm ! 5 lm) with a mobile phase of 5% H2O in MeOH at 1.5 ml/min. The HPLC-MS instrument was operated in selected ion monitoring (SIM)/scan mode. Dinosterol (MW 428) was assigned from the m/z 411 signal resulting from the loss of H2O and addition of a hydrogen atom (M"18+H)+. Fractions were collected in 1 min intervals and three to four fractions were combined on the basis of m/z 411 abundance. A more detailed description of the dinosterol purification and analysis is given by Atwood and Sachs (2012). 2.6. GC–MS identification and quantification Dinosterol in the sterol/alcohol fractions was quantified using GC–MS in SIM mode with dinosterol and heneicosanol calibration standards. Dinosterol calibration standards were prepared by purifying sediment extracts using HPLC, sequentially diluting the stock solution and quantifying with a GC instrument with flame ionization detection (FID). A series of six dinosterol and three heneicosanol quantification standards were prepared that spanned a range of 1–100 ng/ll and 30–300 ng/ll, respectively. After every 10–20 samples, the standards were analyzed and calibration curves calculated to quantify the concentration of dinosterol in each sample. Using this procedure, sub-ng quantities of analyte could be quantified. Samples were derivatized by silylation for identification purposes, since most reference spectra are of the
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II 4α,24-dimethyl-5α-cholestan-3ß-ol
4α-methyl-5α-cholestan-3ß-ol
4α,24-dimethyl-5α-cholest-22E-en-3ß-ol
Detector Response
trimethylsilyl ethers, and by acetylation for quantification. Silylated samples were prepared with 10 ll pyridine and 20 ll bis(trimethylsilyl)trifluoroacetamide (BSTFA) at 60 "C for 1 h. Acetylated samples were prepared with 20 ll pyridine and 20 ll acetic Ac2O at 70 "C for 30 min. A known amount of 5a-cholestane was added as a quantitation standard prior to injection. GC–MS data were acquired with an Agilent 6890 N GC instrument coupled to an Agilent 5975 Mass Selective Detector with either a non-polar Agilent DB-5ms column (60 m ! 0.32 mm i.d. ! 0.25 lm film thickness) or a mid-polarity Varian VF-17 ms FactorFour column (60 m ! 0.32 mm ! 0.25 lm). Samples were injected using a split/splitless inlet in splitless mode at 300 "C. The GC oven program was: 60–150 "C at 15 "C/min, then to 320 "C (held 28 min) at 6 "C/min. The flow of He carrier gas was 1.5 ml/ min. For compound assignment a full scan (m/z 50–700) analysis was performed, while for quantification, selected ions were measured.
35
37
4α-methyl-24-ethyl-5α-cholest-22E-en-3ß-ol 4α,23,24-trimethylcholest-5,22-dien-3ß-ol
A.R. Atwood et al. / Organic Geochemistry 66 (2014) 80–89
IV
V
39
41
VIII VII 43
45
47
Retention Time (min)
3.1. Sedimentary sterol distributions
Fig. 2. Partial GC–MS TIC of 4a-methyl sterol fraction (silylated) from El Junco Lake sediment (124–124.5 cm; VF-17 ms column) following NP-HPLC purification of the sterol/alcohol fraction. See Table 1 and Fig. 3 for names and structures of sterols II-VIII.
El Junco Lake sediment hosts a unique distribution of algal sterols. In the surface sediment, the sterols were dominated by 4a-desmethyl sterols, including cholest-5-en-3b-ol, 5a-cholestan-3b-ol, 24-methylcholest-5-en-3b-ol, 24-methyl-5acholestan-3b-ol, 24-ethylcholesta-5,22E-dien-3b-ol, 24-ethyl-5acholest-22E-en-3b-ol, 24-ethyl-5a-cholesta-7,22E-dien-3b-ol, 24-ethyl-5a-cholestan-3b-ol and 24-ethyl-5a-cholest-7-en-3b-ol (Zhang et al., 2011). In deeper layers the sterol fraction was intermittently dominated by 4a-methyl sterols, particularly 4a,23,24trimethyl-5a-cholest-22E-en-3b-ol (dinosterol; sterol II; Fig. 1). Other 4a-methyl sterols in these layers included 4a-methyl-5acholestan-3b-ol, 4a,24-dimethyl-5a-cholest-22E-en-3b-ol, 4a,24dimethyl-5a -cholestan-3-ol, 4a -methyl-24-ethyl-5a -cholest22E-en-3b-ol, 4 a ,23,24-trimethylcholest-5,22-dien-3b-ol (dehydrodinosterol) and 4a,23,24-trimethyl-5a-cholestan-3-ol (dinostanol; sterol IV; Fig. 2). Due to co-elution of several of the major sterols with dinosterol on the commonly used DB-5ms GC
column (Fig. 1), analysis was generally performed with a VF-17ms column (e.g. Fig. 2; Atwood and Sachs, 2012). A suite of more unusual sterols with a cyclopropyl-containing side chain was found in the layers dominated by the 4a-methyl sterols. These included 22,23-methylene-23,24-dimethyl-5acholestan-3b-ol (gorgostanol; sterol VI; Fig. 1) and its 4a-methyl analogue, 22,23-methylene-4a,23,24-trimethyl-5a-cholestan-3bol (4a-methylgorgostanol; sterol VIII; Fig. 1). Also found was a sterol, the relative retention time (sterol VII; Fig. 1; Table 1) and mass spectrum (Fig. 4A) of which match an unidentified C31 sterol reported in the resting cysts of Peridinium umbonatum var. inaequale, isolated from a Japanese pond (Amo et al., 2010). Based on its similar retention time to other 4a-methyl sterols on NP-HPLC with a Cyano column and a mobile phase of 15% DCM in hexane, we assigned it as a 4a-methyl sterol (Fig. 2; Atwood and Sachs, 2012). As suggested by Amo et al. (2010), the
3. Results and discussion
III 26
II
28
30
Detector Response
32:1 32:2 24 d
V/ 1,ω16 30
30:1 c 27 21 22
29 I
25
31:1
25
27
29
31
33
35
31
73
1,ω18 32
32
IV
ab
1,ω16 30
VI
VII
VIII
39
1,ω18 32
41
43
45
Retention Time (min) Fig. 1. Partial GC–MS TIC of alcohol and sterol fraction (silylated) from El Junco Lake sediment (119–119.5 cm; DB-5ms column). I–VIII: sterols (see Table 1 and Fig. 3 for names and structures), a: 4a-methyl-5a-cholestan-3b-ol, b: 4a,24-dimethyl-5a-cholest-22E-en-3b-ol, c: 24-ethyl-5a-cholestan-3b-ol, d: 4a,23,24-trimethylcholest-5,22dien-3b-ol (dehydrodinosterol), d: n-alkan-1-ols, : n-alken-1-ols, N: diols, .: keto-ols.
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A.R. Atwood et al. / Organic Geochemistry 66 (2014) 80–89 Table 1 GC–MS retention data for sterols (DB-5ms column).
a b c
Compound
Common name
Structurea
MWb
RRTc
23,24-Dimethyl-5a-cholest-22E-en-3b-ol 4a,23,24-Trimethyl-5a-cholest-22E-en-3b-ol 22,23,24-Trimethyl-5a-cholest-22E-en-3b-ol 4a,23,24-Trimethyl-5a-cholestan-3b-ol 4a,22,23,24-Tetramethyl-5a-cholest-22E-en-3b-ol 22,23-Methylene-23,24-dimethylcholestan-3b-ol 22,23-Methylene-4a-methyl-24-ethyl-cholest-5-en-3b-ol 22,23-Methylene-4a,23,24-trimethylcholestan-3b-ol
4a-Desmethyl-dinosterol Dinosterol – Dinostanol – Gorgostanol – 4a-Methylgorgostanol
I II III IV V VI VII VIII
486 500 500 502 514 500 512 514
1.04 1.08 1.08 1.10 1.11 1.12 1.15 1.16
Refer to Fig. 3 for structures. Molecular weight as TMS derivative. Retention time relative to cholesterol TMS.
major ion fragments at m/z 129 and M+"129 are strongly indicative of a D5 sterol (Brooks et al., 1968). However, the major ion fragment at m/z 271 (M+"129–112), in conjunction with a MW of 512, is suggestive of a 22,23-methylene group due to cleavage
(I) 23,24-dimethyl-5α-cholest-22E-en-3β-ol (4α-desmethyl-dinosterol)
of the cyclopropane ring in the side chain, not a D22 sterol as previously suggested. This idea is supported by the fact that 4a-methylgorgostanol and gorgosterol have major ion fragments at M+"112 (Hale et al., 1970; Alam et al., 1979; Fig. 4B). In addition,
(II) 4α,23,24-trimethyl-5α-cholest-22E-en-3β-ol (dinosterol)
(III) 22,23,24-trimethyl-5α-cholest-22E-en-3β-ol
28
21 23 25 20 22 24 18
HO
12 19 11 13 1716 14 15 2 1 10 9 8 34 5 67
29
27
26
(IV) 4α,23,24-trimethyl-5α-cholestan-3β-ol (dinostanol)
HO
HO
HO
(V) 4α,22,23,24-tetramethyl-5α-cholest-22E-en-3β-ol
HO
(VI) 22,23-methylene-23,24-dimethylcholestan-3β-ol (gorgostanol)
HO
(VII) 22,23-methylene-4α-methyl-24-ethyl-cholest-5-en-3β-ol
(VIII) 22,23-methylene-4α,23,24-trimethylcholestan-3β-ol (4α-methylgorgostanol)
HO
HO
(IX) 1,ω20 (1,11) C30 diol OH OH
(X) C30 ω20-keto-1-ol OH O
(XI) C30:0 alkan-1-ol OH
Fig. 3. Structures of compounds identified.
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A.R. Atwood et al. / Organic Geochemistry 66 (2014) 80–89
the fragment at m/z 83 and the lack of one at m/z 69, suggest that the compound has an ethyl group at C-24 (Volkman et al., 1990). We thus tentatively assigned it as 22,23-methylene-4a-methyl24-ethyl-cholest-5-en-3b-ol (sterol VII; Fig. 3), although confirmation requires further investigation. Other unusual sterols included 22,23,24-trimethyl-5a-cholest22E-en-3b-ol (sterol III; Fig. 1). Its identification was based on relative retention time (Table 1) and mass spectrum (Fig. 4C), as it was well separated from other sterols during GC–MS analysis following RP-HPLC of the sterol/alcohol fraction (Fig. 5). To the best of our knowledge, this sterol has only been reported in sediment from Lake Kinneret, Israel (Robinson et al., 1986). In addition, the novel sterol 4a,22,23,24-tetramethyl-5a-cholest-22E-en-3b-ol (sterol V; Fig. 1) was assigned on the basis of its relative retention time during GC–MS (Table 1) and during NP-HPLC (eluting with the other 4a-methyl sterols; Fig. 2), along with its mass spectrum (Fig. 4D), which showed a similar pattern to 22,23,24-trimethyl-5a-cholest-22E-en-3b-ol, but with the addition of 14 mass units (corresponding to a methyl group) to each major fragment ion. A similar comparison could be made between the mass spectra of 4a-desmethyl-dinosterol (Fig. 4E) and dinosterol (Fig. 4F), which also only differ by a 4a-methyl group.
129
Intensity
83
3.2. Sterol sources The 4a-desmethyl sterols that dominated the sterol/alcohol fraction in the surface sediment have been reported in a broad array of microalgae and higher plants (Hartmann and Benveniste, 1987; Volkman et al., 1998; Marshall et al., 2002; Rampen et al., 2010). The C29 sterols with a C24 ethyl group and D7 bond are abundant in many green microalgae as well as some higher plants, while the C29 D5,22-sterols with a C24 ethyl group have been found in higher plants and a variety of algae, including diatoms, raphidophytes and prymnesiophytes (Rˇezanka et al., 1986; Volkman et al., 1990, 1998; Barrett et al., 1995; Marshall et al., 2002; Rampen et al., 2010). In fact, prymnesiophyte algae of the genus Pavlova produce all the major 4a-desmethyl sterols found in the surface sediment, other than the D7 sterols (Volkman et al., 1990). In the layers dominated by 4a-methyl sterols, one of the primary sterols was dinosterol (Fig. 1), which is generally regarded as a biomarker for dinoflagellates (Boon et al., 1979; Volkman, 2003). The other 4a-methyl sterols found in these layers are also produced by dinoflagellates, including 4a-methyl-5a-cholestan-3b-ol, 4a,24-dimethyl-5a-cholest-22E-en-3b-ol, 4a,24-dimethyl-5acholestan-3b-ol, 4a-methyl-24-ethyl-5a-cholest-22E-en-3b-ol, 4 a ,23, 24-trimethylcholesta-5,22E-dien-3b-ol and
75
(A) sterol VII 22,23-methylene-4α-methyl-24-ethyl-cholest-5-en-3β-ol
(B) sterol VIII 4α-methylgorgostanol
M - 129 - 112 271
55
M - 129 383 M+ 512
M - 90 422
50
100
150
200
250
69
300
350
400
450
M - 90 - SC 271 299
M+ 514 M - 15 499
229
500
550
50
100
150
200
69
(C) sterol III 22,23,24-trimethyl-5α-cholest-22E-en-3β-ol
Intensity
M - SC - 2H 359 M - 112 402
250
300
350
400
450
500
550
(D) sterol V 4α,22,23,24-tetramethyl-5α-cholest-22E-en-3β-ol
345 359 257 271 367
374
485 457
50
100
150
200
250
300
350
400
69
500
550
50
Intensity
150
200
250
100
150
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250
300
300
m/z
350
400
450
388
550
50
100
450
514 500
550
359
486 500
400
499 471
(F) sterol II dinosterol
271 471
350
388
69
345
374
100
500
(E) sterol I 4α-desmethyl-dinosterol
257
50
450
381
150
200
250
300
350
400
485
450
500 500
550
m/z
Fig. 4. Mass spectra of sterols (as trimethylsilyl ethers): (A) 22,23-methylene-4a-methyl-24-ethyl-cholest-5-en-3b-ol, (B) 4a-methylgorgostanol, (C) 22,23,24-trimethyl-5acholest-22E-en-3b-ol, (D) 4a,22,23,24-tetramethyl-5a-cholest-22E-en-3b-ol, (E) 4a-desmethyl-dinosterol and (F) dinosterol.
85
III
36
37
38
39
40
41
42
43
44
45
Retention Time (min) Fig. 5. Partial GC–MS TIC of (silylated) dinosterol fraction from El Junco Lake sediment (124–124.5 cm; VF-17 ms column) following RP-HPLC purification of the sterol/alcohol fraction. See Table 1 and Fig. 3 for names and structures of sterols II and III.
4a,23,24-trimethyl-5a-cholestan-3b-ol (Fig. 2; Volkman et al., 1984, 1999b; Mansour et al., 1999). Sterols with a cyclopropyl-containing side chain were also found in the sediment layers dominated by 4a-methyl sterols, including gorgostanol and 4a-methylgorgostanol. In particular, 4a-methylgorgostanol is a highly source-specific biomarker, having been reported only in three asymbiotic species of dinoflagellates: Peridinium foliaceum (Alam et al., 1979; Withers et al., 1979), Alexandrium monilatum (Wengrovitz et al., 1981) and Alexandrium tamarense (Piretti et al., 1997), and in soft coral with symbiotic zooxanthellae (Kobayashi et al., 1982; Kokke et al., 1982). Of the few organisms found to produce 4a-methylgorgostanol, only dinoflagellates of the genus Peridinium are likely to exist in El Junco Lake. The zooxanthellae of the Caribbean gorgonians are marine species (Kokke et al., 1981), while the dinoflagellates of the genus Alexandrium are found in marine and estuarine environments. Alexandrium tamarense is a dinoflagellate that can produce paralytic shellfish-toxins and has been found in subarctic to tropical coastal and estuarine waters (Lilly et al., 2007). In culture studies, it was found to have an optimal growth rate at a salinity ca. 20–30, while it did not survive at a salinity of 10 and below (Lim and Ogata, 2005). Similarly, Alexandrium monilatum is a toxic red tide dinoflagellate found in coastal and estuarine waters in subtropical and tropical regions (May et al., 2010). In culture studies, it exhibited low growth rate below a salinity of 15 (Juhl, 2005). Because of the salinity-dependent growth rate of these organisms, dinoflagellates of the genus Alexandrium are unlikely to be present in the freshwater El Junco Lake. In contrast, Peridinium is primarily a freshwater dinoflagellate genus with some brackish and marine species (Trigueros et al., 2000). P. foliaceum, the species found to produce 4a-methylgorgostanol, is an autotrophic non-toxic red tide dinoflagellate found in marine and estuarine systems, primarily in the subtropics. It can tolerate a wide range of temperature (12–25 "C) and salinity (0.2– 31 ppt; Trigueros et al., 2000). The major sterols reported in P. foliaceum (Withers et al., 1979) were also found in El Junco Lake sediment, including dinosterol, 4a,24-dimethyl-5a-cholestan-3b-ol, cholest-5-en-3b-ol (cholesterol), and 24-methylcholest-5-en-3bol, with minor amounts of gorgostanol and 4a-methylgorgostanol in the sediment. To the best of our knowledge, only one study has reported the existence of 4a-methylgorgostanol in a freshwater environment – subtropical Lake Kinneret in Israel (Robinson et al., 1986). The sedimentary 4a-methyl sterol composition of that lake closely
matches that of El Junco Lake, with the primary 4a-methyl sterols being dinosterol, 4a,23,24-trimethylcholest-5,22-dien-3b-ol (dehydrodinosterol) and dinostanol, and the minor constituents 4a-methyl-5a-cholestan-3b-ol, 4a,24-dimethyl-5a-cholestan-3bol and 4a-methylgorgostanol (Fig. 2). Peridinium cintum var. gatunense was the predominant dinoflagellate in a multi-year study of Lake Kinneret (Viner-Mozzini et al., 2003). Although 4a-methylgorgostanol was not found in P. gatunense isolated from that setting, the authors hypothesized that 4a-methylgorgostanol may be produced by P. gatunense under environmental conditions different from those that characterized the sampling period (Robinson et al., 1986). As P. gatunense has been found to produce resting cysts (Viner-Mozzini et al., 2003) and the sterol composition of resting cysts can substantially differ from that of the motile cells (Amo et al., 2010), it is possible that 4a-methylgorgostanol is produced by P. gatenense during encystment. The sedimentary distribution of 4a-methylgorgostanol, dinosterol and 4a,22,23,24-tetramethyl-5a-cholest-22-en-3b-ol suggests that these compounds were derived from the same source. Through the 3.7 m of sediment, the concentration of dinosterol and 4a,22,23,24-tetramethyl-5a-cholest-22-en-3b-ol covaried with 4a-methylgorgostanol (r2 0.91 and 0.92, respectively; p < 0.05; Fig. 6). The results strongly suggest that dinosterol was produced by a dinoflagellate of the genus Peridinium throughout the sediment record. Although the concentrations of 22,23-methylene-4a-methyl-24-ethyl-cholest-5-en-3b-ol and 22,23,24-trimethyl-5a-cholest-22E-en-3b-ol were not quantified (due to the low abundance of 22,23-methylene-4a-methyl-24-ethyl-cholest5-en-3b-ol and the fact that the GC–MS analysis did not include ions from 22,23,24-trimethyl-5a-cholest-22E-en-3b-ol during SIM), the co-existence of these compounds and the rarity of their cyclopropyl- and 22,23,24-trimethyl-containing side chains suggests that they also originated from the same organism as 4a-methylgorgostanol and 4a,22,23,24-tetramethyl-5a-cholest22-en-3b-ol. In particular, the sedimentary profiles of these sterols suggest that the two 22,23,24-methyl sterols are not diagenetic products of their cyclopropyl-containing side chain counterparts. Although sterols with 22,23,24-trimethyl-containing side chains have been reported in organisms (Kerr et al., 1999), it has been proposed that sedimentary 22,23,24-methyl sterols are produced through the opening of the cyclopropyl ring during diagenesis (Thomas et al., 1993; Hou et al., 1999, 2000; Rampen et al., 2009). However, in El Junco Lake sediment, the linear relationship between 4a,22,23,24-tetramethyl-5a-cholest-22-en-3b-ol and
700
Concentration (µg/g dry sediment)
35
II
C32 1,ω18-diol
Detector Response
24-ethylcholestan-3ß-ol
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600
dinosterol r 2 = 0.91
500 400 300 200
4α,22,23,24-tetramethyl sterol r 2 = 0.92
100 0 0
2
4
6
8
10
12
14
16
18
20
4α-Methylgorgostanol Concentration (µg/g dry sediment) Fig. 6. Co-variation of dinosterol and 4a,22,23,24-tetramethyl-5a-cholest-22E-en3b-ol with 4a-methylgorgostanol in El Junco Lake sediment (0–372 cm).
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4a-methylgorgostanol (Fig. 6), the lack of any discernable increase in the ratio of 4a,22,23,24-tetramethyl-5a-cholest-22-en-3b-ol to 4a-methylgorgostanol concentration with depth (Fig. 7) and the absence of the saturated analogues of the 22,23,24-methyl sterols suggests that the 22,23,24-methyl sterols are produced by a dinoflagellate of the genus Peridinium, not through sedimentary diagenesis of 4a-methylgorgostanol and gorgostanol. Finally, evidence from a nearby lake supports our hypothesis that dinosterol and the unusual sterols in El Junco Lake are produced by a dinoflagellate from the genus Peridinium. Botos Lake is a freshwater crater lake in Costa Rica with similar characteristics to El Junco Lake. It is acidic (pH 4.4), oligotrophic and alpine (2600 m), with a maximum depth of 9 m and a well-mixed water column (Umaña-Villalobos, 2001). In Botos Lake, Peridinium inconspicuum dominated the phytoplankton community throughout a 2 yr sampling period (Umaña-Villalobos, 2001). Indeed, this dinoflagellate has been found in a number of lakes in Central America and has been referred to as the dominant tropical dinoflagellate (Umaña-Villalobos, 2001). Based on the available evidence, we conclude that the primary source of dinosterol in El Junco Lake is likely a dinoflagellate from the genus Peridinium, possibly P. inconspicuum, P. gatunense, or P. foliaceum and that this source has not changed throughout the sediment record. The evidence includes the fact that a dinoflagellate from the genus Peridinium exists in similar environments to El Junco Lake, produces the unusual 4a-methylgorgostanol and 22,23methylene-4a-methyl-24-ethyl-cholest-5-en-3b-ol found in El Junco Lake sediment and dominates Lake Kinneret (from which the sole report of 22,23,24-trimethyl-5a-cholest-22-en-3b-ol, also found in El Junco Lake sediment, is derived). In addition to being highly source-specific in this environment, dinosterol exists in abundance throughout much of the 372 cm of recovered sediment (1–678 lg/g dry sediment), making it an excellent target for isotopic paleoclimate applications. 3.3. Sedimentary diols, triols, keto-ols and n-alkenols Long chain alkyl diols, triols, keto-ols and n-alkenols exist in abundance in El Junco Lake sediment and have the potential to yield additional paleoclimate information if their sources are determined. At different depths in the sediment, the dominant diols alternated between C29 9,10-diol, C30 1,x16-diol, C30 1,x18diol, C30 1,x20-diol, C32 1,x18-diol, and/or C32 1,x20-diol, while the corresponding keto-ols tended to coexist with the C30 and C32 diols (Fig. 1; Zhang et al., 2011). The C29 1,x9,x10-triol was also found in high abundance in many sediment intervals, as were 0 50
Depth (cm)
100 150 200
C30-C32 alken-1-ols with one and two degrees of unsaturation (Fig. 1; Zhang et al., 2011). Long chain alkyl diols and keto-ols have been reported in a variety of organisms, ranging from higher plants to aquatic algae. However, different organisms have distinctive signatures with respect to the isomers of the diols and keto-ols that they produce (Jetter and Riederer, 1999; Volkman et al., 1999a; Méjanelle et al., 2003; Speelman et al., 2009). Expanding upon the identification of these compounds in El Junco Lake sediment made by Zhang et al. (2011), here we have identified the specific sources of the diols, triols, keto-ols and n-alkenols. The C30–C32 1,x20-diols and keto-ols, C29 9,10-diol, and C29 1,x9,x10-triol have been reported in Azolla filliculoides, an aquatic fern found throughout tropical and temperate freshwater ecosystems (Speelman et al., 2009), Osmunda regalis, a terrestrial fern found in temperate regions (Jetter and Riederer, 1999), and several species of the poppy family, Papaveraceae (Jetter et al., 1996). However, these compounds have not been reported in aquatic algae. In contrast to the 1,x20 isomers of C30 and C32 diol and keto-ol, the 1,x16 and 1,x18 isomers (with the exception of C32 x16-keto1-ol) have been reported in algae of the class Eustigmatophyceae (Volkman et al., 1992, 1999a; Méjanelle et al., 2003). In addition, the predominant n-alken-1-ols in the sediment, namely C30:1, C31:1, C32:1 and C32:2 n-alken-1-ols (Fig. 1), also appear to have an algal source, as reports of such long chain n-alkenols have been limited to several types of aquatic algae, including eustigmatophytes (Volkman et al., 1992; Méjanelle et al., 2003), chlorophytes (Allard and Templier, 2000) and marine haptophytes (Rontani et al., 2004). 3.4. Diols, triols and keto-ols in El Junco Lake vegetation samples Due to the abundance of diols, triols, and keto-ols in the sediment, we investigated the sources of these compounds in the surrounding vegetation in order to evaluate their source specificity and therefore suitability as indicators of paleoenvironmental change. Samples of twelve native terrestrial and aquatic plant species were analyzed for this purpose. Of these, only the two ferns (Azolla microphylla and Cyathea weatherbyana) were found to contain diols, triols, and/or keto-ols. A. microphylla, an aquatic fern that populates the shoreline, was found to contain C30–C33 terminal/mid-chain diols and C27–C30 vicinal diols (Fig. 8A). All the terminal/mid-chain diols except C30 had only one isomer present – that with the mid-chain OH at the x20 position (Table 2). For the C30 terminal/mid-chain diol, both the 1,x20 and 1,x18 isomers were present. In contrast, all the vicinal diols were found to contain OH groups at C-9,10 positions (x18– x22). The C29 1,x9,x10-triol was also found to be a major constituent of the TLE from A. microphylla. A series of mid-chain n-alkanols with the OH at the x20 position and an odd number of carbons ranging from C29 to C33 were also found in the TLE. Cyathea weatherbyana, an abundant tree fern around the lake, was found to contain several long chain keto-ols. They consisted of C30 x20-keto-1-ol (structure X in Fig. 3), C30 x18-keto-1-ol and C32 x20-keto-1-ol (Fig. 8B; Table 2). 3.5. Sources of sedimentary diols, triols, keto-ols and n-alkenols
250 300 350 400 0
2
4
6
8
10
12
[4α,22,23,24-tetramethyl sterol] / [4α-methylgorgostanol] Fig. 7. Ratio of concentration of 4a,22,23,24-tetramethyl-5a-cholest-22E-en-3b-ol to 4a-methylgorgostanol with depth in El Junco Lake sediment.
The lipid compositions of the vegetation samples were consistent with the ferns surrounding the lake (A. microphylla and C. weatherbyana) being the sources of the abundant sedimentary C30 and C32 1,x20-diols and keto-ols, C29 9,10-diol, and C29 1,x9,x10-triol. It is also possible that the sedimentary keto-ols were produced through diagenetic alteration of the corresponding diols (Ferreira et al., 2001). In addition, we conclude that the sources of the C30 1,x16-diol and keto-ol, C32 1,x18-diol and keto-ol and C30–C32 n-alken-1-ols in El Junco Lake sediment are
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32
Detector Response
(A)
9,10 29 c 29
9,10 27
9,10 28 a
42
C29 triol 31
9,10 30
31
d
b
33
33
30
46
50
54
58
54
58
Detector Response
(B) fernene cd
b
42
32
30 e
h
h
h
46
50
Retention Time (min) Fig. 8. Partial GC–MS TIC of the neutral fraction of two El Junco Lake vegetation samples: (A) Azolla microphylla and (B) Cyathea weatherbyana. a: cholest-5-en-3b-ol (cholesterol), b: 24-methylcholest-5-en-3b-ol (campesterol), c: 24-ethylcholest-5-en-3b-ol (b-sitosterol), d: 24-ethylcholestan-3b-ol (stigmastanol), e: 24-methylcholestan3b-ol (campestanol), h: hopenoids, N: diols, .: keto-ols, s: sec-alkanols.
Table 2 Distribution of diols, keto-ols, triols, and alkanols in El Junco Lake ferns. Chain length
A. microphylla Terminal/mid-chain diols
C27 C28 C29 C30 C31 C32 C33
1,x20 (100); 1,x18 (53)a 1,x20 1,x20 1,x20
C. weatherbyana Vicinal diols
x18,x19 x19,x20 x20,x21 x21,x22
Triols
sec-Alkanols
1,x9,x10
x20
Keto-ols
1,x20 (100); 1,x18 (66)a
x20 1,x20
x20
a The relative proportion of isomers (in parentheses) was estimated from the relative intensity of the fragment ions resulting from cleavage of the CAC bond adjacent to the mid-chain OTMS group.
likely restricted to aquatic algae, as we found no evidence for these compounds in any of the El Junco Lake vegetation samples. However, the C30 1,x18-diol and keto-ol appear to be less source-specific as, in addition to a eustigmatophyte source, we found them in the El Junco ferns A. microphylla and C. weatherbyana, respectively. Because the C30 and C32 1,x20-diol and keto-ol, C29 9,10-diol and C29 1,x9,x10-triol are attributed to production from terrestrial and shoreline ferns, while the C30 1,x16-diol and keto-ol, C32 1,x18-diol and keto-ol and C30–C32 n-alken-1-ols are attributed to aquatic algae, changes in the relative abundances of these biomarkers can be used to estimate past changes in lake hydrology. While the terminal/mid-chain diols and keto-ols do not make good targets for isotope analysis in this environment due to the variety of isomers that co-elute during GC analysis, the vicinal diols and triols do not seem to have these coelution issues. This feature, in conjunction with their source specificity (A. microphylla), may make them suitable targets for isotope analysis, if they are of suf-
ficient abundance through the sediment record. In this way, these biomarkers can provide additional tools with which to reconstruct past environmental and climatic change in El Junco Lake.
4. Conclusions A variety of sterols, diols, triols, keto-ols and n-alkenols in El Junco Lake sediments was characterized in order to identify potential biomarkers for use in paleoclimate applications. A suite of highly unusual sterols was found, including 22,23-methylene23,24-dimethyl-5a-cholestan-3b-ol (gorgostanol), 22,23-methylene-4a,23,24-trimethyl-5a-cholestan-3b-ol (4a-methylgorgostanol) and 22,23,24-trimethyl-5a-cholest-22-en-3b-ol. In addition, two novel sterols were tentatively assigned as 4a,22,23,24-tetramethyl-5a-cholest-22-en-3b-ol and 22,23-methylene-4a-methyl24-ethyl-cholest-5-en-3b-ol. The similarity in the 4a-methyl sterol composition between dinoflagellates from the genus Peridinium
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and the lake sediment, in conjunction with the presence of the unusual sterols 4a-methylgorgostanol, 22,23,24-trimethyl-5acholest-22-en-3b-ol, and the tentatively assigned 22,23-methylene-4a-methyl-24-ethyl-cholest-5-en-3b-ol, strongly suggests that a dinoflagellate from the genus Peridinium is the dominant source of these sterols in the lake. In addition, we conclude that dinosterol and 4a,22,23,24-tetramethyl-5a-cholest-22-en-3b-ol, in addition to the above sterols, was produced by the same species of dinoflagellate throughout the sediment record due to the strong correlation between the concentration of dinosterol and 4a-methylgorgostanol (r2 0.91) and between the concentration of 4a,22,23,24-tetramethyl-5a-cholest-22-en-3b-ol and 4a-methylgorgostanol (r2 0.92). The sedimentary profiles of these sterols suggest that the two 22,23,24-methyl sterols are not diagenetic products of their cyclopropyl-containing side chain counterparts. Knowledge of their biological sources enables their use as biomarkers to study past changes in the lake hydrology and regional climate. In particular, the fact that dinosterol was relatively abundant throughout most of the sediment profile (1–678 lg/g dry sediment) and highly source-specific in this environment makes it a valuable target for paleoclimate reconstruction based on changes in its abundance and hydrogen isotope ratio values. In addition to the sedimentary sterols, sources of the abundant sedimentary long chain alkyl diols, triols, keto-ols and n-alkenols were evaluated. The C30 and C32 1,x20-diols and x20-keto-1-ols, C29 9,10-diol, and C29 1,x9,x10-triol found in the lake sediment were identified in the ferns Azolla microphylla and Cyathea weatherbyana. The absence of reported sources of these compounds outside the fern (and poppy) family leads us to conclude that the sources of these compounds are likely limited to these ferns. In contrast, sources of the C30 1,x16-diol and x16-keto-1-ol, C32 1,x18-diol and x18-keto-1-ol, and the C30–C32 n-alken-1-ols, which were not found in the lake vegetation samples, are likely limited to aquatic algae. Due to the disparate sources of these biomarkers, their relative distribution in the sediment record presents another tool with which to study past environmental change in El Junco Lake. Acknowledgments This study was based upon work supported by the U.S. National Science Foundation under Grants EAR-0823503 and ESH0639640and the U.S. National Oceanic and Atmospheric Administration under Grant No. NA08OAR4310685 to J.S. A.A. was supported by the National Science Foundation Graduate Research Fellowship Program and the Department of Energy Global Change Education Program. The authors would like to thank R. Smittenberg and K. Ricke for preparation and GC-MS analysis of vegetation samples – initial analyses of the vegetation samples can be found in Ricke (2004). We would also like to thank Z. Zhang for sample preparation and O. Kawka and J. Gregersen for support in the lab. We acknowledge P. Colinvaux for bringing El Junco Lake to our attention and the following for assistance in the field and logistical support: P. Colinvaux, J. Overpeck, M. Steinitz-Kannan, J. Conroy, R. Smittenberg, the Galápagos National Park and the Charles Darwin Research Station. Finally, we thank S. Wakeham and I. Bull for useful discussions, as well as S. Rampen and M. Amo for thoughtful reviews. Associate Editor—E.A. Canuel References Alam, M., Ray, S.M., Martin, G.E., 1979. Dinoflagellate sterols. 2. Isolation and structure of 4-methylgorgostanol from the dinoflagellate Glenodinium foliaceum. Journal of Organic Chemistry 44, 4466–4467.
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