Soil Perturbation in Mediterranean Ecosystems Reflected by

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Agricultural and Environmental Chemistry

Soil Perturbation in Mediterranean Ecosystems Reflected by Differences in Free Lipid Biomarker Assemblages Pilar Tinoco, Gonzalo Almendros, and Jesús Sanz J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01483 • Publication Date (Web): 04 Sep 2018 Downloaded from http://pubs.acs.org on September 5, 2018

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Journal of Agricultural and Food Chemistry

== AGRICULTURAL AND ENVIRONMENTAL CHEMISTRY ==

RESEARCH ARTICLE:

RUNNING TITLE: Soils lipid signature

Soil Perturbation in Mediterranean Ecosystems Reflected by Differences in Free Lipid Biomarker Assemblages

†

‡*

§

PILAR TINOCO , GONZALO ALMENDROS , JESÚS SANZ

†

Universidad Alfonso X el Sabio, Campus de Villanueva de la Cañada. Av. Universidad 1, E-28691 Madrid ‡

§

MNCN, CSIC, Serrano 115B, 28006-Madrid, Spain

Instituto de Química Orgánica General, CSIC, Juan de la Cierva 3, E-28006 Madrid, Spain

*

Corresponding author. E-mail: [email protected] (G. Almendros)

1

ACS Paragon Plus Environment



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1

Environmental information provided by free lipids in soil samples collected from control and

2

disturbed plots (Madrid, Spain), was assessed by comparing molecular assemblages of terpenoids

3

and distribution patterns of alkanes and fatty acids (FAs), analyzed by gas chromatography-mass

4

spectrometry (GC-MS). Wildfires in pine forests led to increased proportions of retene,

5

dehydroabietin and simonellite. Friedo-oleananes were characteristic in soils under angiosperms,

6

and norambreinolide-type diterpenes in soils encroached by Cistus bushes. Steroids were major

7

compounds in pastured site. Enhanced Shannon’s lipid biodiversity index in disturbed soils

8

compared to control soils suggested patterns of recent lipids overlapped with a preserved original

9

lipid signature. The extent of the environmental impacts was illustrated as Euclidean distances

10

between paired control and disturbed sites calculated using as descriptors the compounds in alkyl

11

homologous series. As expected, reforestation, bush encroachment, wildfires and cultivation were

12

reflected by changes in the molecular record of lipids in soils.

13 14 15 16

KEYWORDS: Alkane, Biomarker, Terpene, Fatty acid, Molecular tracer, Signature lipid

17

1



18

INTRODUCTION

19

The lipid fraction of soil is often considered a valuable source of environmental information,

20

representing a continuous molecular record shedding light on climate change and the intensity of

21

organic matter (OM) turnover1–4. In fact, whereas the soil lipid fraction originally consists of a

22

heterogeneous molecular assemblage inherited mainly from plants and microorganisms5,6, further

23

abiotic or microbial transformation of these biogenic lipids provides additional compounds in the

24

soil7–11. The dynamics of soil lipids is complex since, apart from the above processes, lipid mixtures

25

are subjected to continuous biodegradation of their comparatively labile molecules. Nevertheless,

26

lipid molecules can also be included into organo-mineral structures in progressively transformed

27

soil organic matter pools12–14 with an improved preservation of compounds associated with small

28

size aggregate fractions15. Other fractions of lipids present in soil, which require chemical

29

treatments for their release in the form of free molecules, may correspond both to cellular

30

constituents in not still degraded biomass, and to condensation products incorporated to humic

31

substances. Therefore, the balance between biodegradation and humification processes could be

32

monitored by the minor but diagnostic fraction of lipids temporarily free in the soil, which are the

33

subject of this study. Hence, the surviving lipids which can be directly isolated from soil in the form

34

of free compounds could represent a molecular signature for reconstructing recent and past soil

35

processes, since their occurrence could in most cases depend on the environmental impact on

36

terrestrial ecosystems16–19.

37

Finally, specific soil lipids are also important due to their role on soil processes, acting as

38

antimicrobial agents20–23 or in the alellopathic interaction between higher plants1,24,25; or in insect-

39

host plant relationships 26 as well as through the effect on soil physical properties, mainly

40

aggregation and soil water repellence27–30.

41

In the present study, the aim was the analytical comparison between free soil lipids from relict

42

forests and those from soils in adjacent sites under the same climatic conditions and the same

43

original geological substrate, but affected by environmental perturbation processes typical of 2


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continental Mediterranean ecosystems in central Spain. Both individual signature compounds, as

45

well as the distribution patterns of the major series of alkyl compounds, were analysed in 16 well

46

characterised ecosystems in central Spain, consisting of control forests or sites affected by (i)

47

clearing and bush encroachment, (ii) cultivation, (iii) wildfire and (iv) reforestation with pine. The

48

specific objectives would be to assess the extent of the biogeochemical changes undergone by the

49

soils due to the environmental perturbation, to test the response of the biomarker assemblages to

50

environmental changes and to compare hypothetical new proxies for these perturbations.

51 52

MATERIAL AND METHODS

53 54 55

Sampling Soil samples from 8 contrasting continental Mediterranean forest ecosystems in Madrid (central

56

Spain) representative for sclerophyllic (oak), mesophylic (chestnut and ash) or pine forests were

57

collected. In addition to the control sites, another set consisting of 8 altered neighbor soils with the

58

same climatic, geologic and topographic features were sampled31. A two character labelling code

59

was used to refer to the sampling sites: the first letter (see below) was the code for the soil

60

series, followed by an odd number in the case of the relict ecosystems and an even number for

61

the perturbed ones. The distance between sampling points in the paired soils always was < 100 m.

62

Some general features of the soil samples are listed in Table 1.

63

Sampling was carried out in duplicate between April and June: samples were collected after

64

removing the litter layer, when existing, and the soil material (the whole O horizon) was collected

65

with a spade. In order to obtain representative samples averaging the possible spatial variability in

66

the plots, composite samples were taken from each plot. Each individual sample was prepared by

67

mixing three soil subsamples of ca. 1 L from the points of a virtual triangle of ca. 100 m side. The

68

samples were air-dried, litter and root fragments were hand-picked and the resulting material was

69

sieved to 2 mm and used to determine routine analytical characteristics, calculating the least 3



70

significant difference illustrating spatial variability in soil taxonomic characteristics. A combined

71

subsample for the molecular characterization of the lipid fraction was prepared by mixing the

72

subsamples.

73

The characteristics of the 16 ecosystems (8 control sites, coded with a label including an odd

74

numeral + 8 disturbed sites, coded with even numerals) are shown in Table 2. Series R corresponds

75

to soil areas covered by the original oak forest (Quercus ilex subsp. ballota: samples R1 and R3). At

76

some places, the oak had been removed and the site reforested with Pinus pinea or Pinus pinaster

77

(R2 and R4, respectively). The series of cleared forests corresponded to oak (C1) or ash (Fraxinus

78

angustifolia, C3) forest dedicated to cereal cultivation (C2) or grazing pasture (C4). In other cases,

79

oak forests were cleared in a historical period for wood extraction (B1, B3), then occupied by

80

Mediterranean bush with Cistus (B2) or Cytisus (B4). The effect of wildfire was examined by

81

comparing forests of Pinus halepensis (F1) or Pinus sylvestris (F3) with the adjacent sites affected

82

by high intensity (F2) or medium intensity (F4) forest fires.

83 84

Lipid analysis

85

Soil samples (50-g) were Soxhlet extracted with petroleum ether (40–60 °C) for 24 h. This

86

solvent was chosen in order to prevent the removal of dark-colored nonvolatile macromolecular

87

material, probably oligomers of humic substances or lignins, that is co-extracted when using more

88

polar solvents3. Preliminary experiments using polar solvents such as CH2Cl2-MeOH produced, in

89

the case of forest soils, very dark brown extracts not suitable for direct GC analysis. The extract was

90

filtered and concentrated in a rotary evaporator to approximately 50 mL, dried under a stream of

91

N2 at room temperature (20–25 °C) and the residue weighed and methylated with CH2N2/Et2O.

92

The lipids were separated and identified using GC-mass spectrometry (GC-MS) with an HP 5890

93

gas-chromatograph connected to an HP 5971 mass detector (EI, 70 eV). A cross-linked OV-1

94

column (25 m × 0.25 mm i.d. × 0.25 µm film thickness) with and He flow of 1 mL min-1 was used.

95

Constant pressure, and split injection were used. The oven temperature was programmed from 70 4


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-1

96

°C to 320 °C (held 20 min) at 4 °C min . Injection temperature was 300 °C. Some splitless

97

injections were also carried out for trace compound identifications; no significant differences in

98

quantitative results were found between both injection techniques. Total ion current (TIC) areas

99

(acquisition range 39–530 amu) were used for quantitative measurements. Compounds were

100

assigned from their electron impact mass spectra and confirmed, when possible, by comparison

101

with those in the spectral databases or with standards of the authentic compounds.

102

Identification was confirmed by using retention indices calculated for all the compounds from

103

their retention times and those of the n-alkane series, in programmed-temperature conditions.

104

Coelution caused overlapped peaks within FA methyl ester and alkanol series, which were

105

determined from their single ion characteristic traces using the ions at m/z 74 and 69,

106

respectively. Integration values of the peaks in the traces for these single ions were transformed

107

into total ion counts by using suitable correction factors that were calculated from the

108

fragmentation patterns of pure compounds32.

109 110 111

RESULTS and DISCUSSION The total lipid concentration ranged between 0.05 and 9.50 g kg-1 (Table 2). Fig. 1 illustrates the

112

composition of the petroleum ether extract from some representative samples. About 100

113

compounds in each sample, mainly n-alkanes, n-alkanols and FAs were tentatively assigned (Table

114

3). Depending on the soil, variable proportions of cyclic compounds mainly monoterpenes,

115

sesquiterpenes, diterpenes, triterpenoids and steroids, were present (Figs. 2 and 3).

116

Many of the identified compounds could be considered as typical biomarkers, to the extent

117

that they have definitive chemical structures, which can be related directly or indirectly through a

118

set of structural alterations to biogenic sources, and they cannot be synthesized by abiogenic

119

processes7. Typically, monoterpenes and cyclic diterpenes (the latter as hydrocarbons, acids,

120

alcohols, or other derivatives) are major constituents of gymnosperms resins33–40. Other

121

compounds as sterols may derive mainly from the cell walls of animals and plants whereas 5



122

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37

triterpenoids are widely distributed in the epicuticular constituents of higher plants .

123 124

Cyclic compounds

125

Table 4 and Fig. 2 show the compounds with a terpenoid structure. They amounted to between

126

0.9 and 51.9% as proportions of the total ion chromatographic area in the different samples.

127

Cyclic diterpenes (as hydrocarbons, acids, alcohols, or other derivatives) were the most

128

abundant cyclic compounds (0.9–45.2%, Table 5) and belonged to three families: abietane,

129

pimarane and labdane (Fig. 2). The abietane-type diterpenoids were the most abundant, mainly

130

in samples derived from Pinus pinea (R2: 22.3%) and Pinus halepensis (F1: 25.3%).

131

Dehydroabietic acid (compound 95, Table 4), was the most frequent diterpenoid acid in all

132

samples. In fact, this compound is the resin acid most common in the geosphere where it

133

originates from the rapid conversion of abietic acid41–42.

134

There was also an appreciable abundance of abietic acid (compound 101), occurring in

135

samples R2 and F1, and levopimaric acid (99) in samples R2, F1 and F4. Seco-dehydroabietic

136

acid33 which appeared as two isomers (1α– and 2β–) was frequent in the soil lipids under pine

137

forest. In addition, samples R2, R4, F1 and F4 had appreciable proportions of alteration products

138

of dehydroabietic acid, such as 7-oxodehydroabietic (110), 7-hydroxydehydroabietic (111) and

139

15-hydroxydehydroabietic acids (112).

140

A series of abietane hydrocarbons, also presumably derived from the above acids, was also

141

present. The highest proportion of these hydrocarbons corresponded to lipids from soils under

142

burned forest, F2. Dehydroabietin was present in all the soils under pine43 but other typical

143

hydrocarbons, formed by transformation of resin acids8, were also present, such as

144

dehydroabietane (58), 19-norabieta-4,8,11,13-tetraene (40), simonellite (37) and retene (31).

145 146

The pimarane-type diterpenoids (Fig. 2) are traditionally considered to be less stable than the abietanes and, consequently, were present in lower proportions, between 0.1 and 18.5% (Table

6


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147

5). Pimaric acid (96), was frequent in all samples under pine forest, but occurred only as traces in

148

samples C3, C4 and B4.

149

Sandaracopimaric (98), 8,15-isopimaradien-18-oic acid (97) and isopimaric (101) acids were

150

identified. The latter was in high proportion (5.9%) in R2, under Pinus pinea. The labdane

151

diterpenes (Fig. 2) were not abundant (0.1–4.8%) appearing only in R4, B4, F1, F3 and F4. The

152

highest proportion of labdanes occurred in B4, under Cistus bush. This sample also contained

153

eperuic acid (104), cativic acid (105), labdanolic acid (113) and norambreinolid (36). The major

154

compound was labdanolic acid (1.4%). Other labdane-type diterpenoids present in soils under

155

pine were manoyl oxyde (73), pinifolic acid (126) and anticopalic acid (103).

156

As expected, the highest proportion of the above resin acids occurred in the soils under pine

157

(R2, R4, F1, F2, F3 and F4). The presence of small relative amounts of these compounds in C3

158

(ash forest), C4 (pasture) and B2 (Spanish broom) could be explained by wind transport of

159

aerosols from adjacent pine forests, which would contribute to the fact that terpenoids are likely

160

over represented in the sedimentary record43. Nevertheless, "classical" conifer biomarkers of the

161

abietane series have also been reported to be widespread in several cyanobacterial strains44. In

162

any case, in environmental conditions abietanes readily turn into dehydroabietic acid, and have

163

no tendency to accumulate in the advanced transformation stages of soil lipids3.

164

Decarboxylation and aromatization are considered to be trends during ageing of resin acids8

165

leading to diterpene hydrocarbons at the final transformation stages. These hydrocarbons, with

166

the highest proportion in F2, would reflect the effect of fires in producing decarboxylation and

167

aromatization reactions45,46.

168

The fire-affected soils showed decreased proportions of monoterpenes, sesquiterpenes and

169

diterpenoids and concomitantly increased proportions of diterpene hydrocarbons. In fact,

170

wildfires in pine forests would lead to thermal dehydrogenation and decarboxylation of

171

diterpene resin acids47 resulting into aromatic hydrocarbons such as retene, dehydroabietin and

172

simonellite. Sample F4 showed an increased proportion of diterpenes (Table 5), as could 7



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45

173

correspond to thermal distillation of plant waste incorporated into soil . In particular

174

sesquiterpene patterns were quite responsive to the intensity of the fire (F2 > F4).

175

Triterpenoids (2.1–3.9% of the total chromatographic area) were present in R4, C4, B1, B4, F2

176

and F4 (Table 5 and Fig. 2). This group included typical sterols (27, 28 or 29 C atoms). Sample C4

177

displayed the greatest number of sterols, the main ones being cholesterol, campesterol and β-

178

stigmasterol. In R4 the major sterol was stigmast-5-en-3β-ol and in F4 a steroidal ketone

179

(stigmast-4-en-3-one) was found. Other triterpenoids had a pentacyclic skeleton34,35. Friedelan-

180

3-one (friedelin) was detected (Fig. 2) in B1 and B4, whereas friedo-olean-14-en-3-one and

181

ursolic acid were found in F2.

182

Mainly F1 (Pinus halepensis) contained monoterpenes (4.7%); α-pinene and β-pinene, being

183

the most abundant (2% each). Myrcene, γ-terpinene, terpinolene, α-terpinene and p-cymenol

184

(Table 4, Figs. 1 and 2) were also identified. Several sesquiterpenes were also present in F1

185

(Table 4, Fig. 2), mainly caryophyllene (14, 0.4%) and caryophyllene oxide (23, 1.1%). Other

186

sesquiterpenes present in lower proportions were cadalene, calamenene, α-humulene, α-

187

cubebene, α-copaene, α-muurolene, δ-cadinene, β-eudesmol and cembrene (9, 13, 15–19, 24

188

and 59, respectively). No sesquiterpenes were found in other samples, although the lipid

189

fraction of R4 (Pinus pinaster) included a trace of cadalene (0.1%). The fact that monoterpenes

190

were not major compounds in the samples may be explained by the fact that monoterpenes

191

have a relatively low boiling point and probably they are not strongly retained in Mediterranean

192

soils48. In addition, the presence of oxygen-containing functional groups as well as unsaturated

193

bonds in lipid molecules makes lipids suitable for strong retention in the soil organo-mineral

194

matrix, including physical occlusion and hydrophobic interactions3. In active terrestrial

195

ecosystems the occurrence and intensity of such processes should also be related to the

196

selective preservation of free lipid assemblages.

197

Concerning sterols, the dominance of cholesterol, of animal origin, in the pastured site C4 agree

198

with the current use of this soil. 8


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199


Finally, in two samples (R4 and B1) there were detectable proportions of DDE (1,1,1-trichloro-

200

2,2-bis(p-chlorophenyl)ethylene), derived from the degradation of

201

dichlorodiphenyltrichloroethane (DDT).

202 203

Alkyl series

204

Figure 1 and Table 3 show that the major lipid compounds corresponded to alkyl compounds:

205

alkanes (0.5–68.1%), FAs (4.9–55.9%) and alkanols (5.0–53.5%). Fig. 3 illustrates some distribution

206

patterns (Cno vs. relative abundance) of the n-chain homologues of the major series.

207

Homologous series of n-alkanes, n-alkanols and n-alkanoic acids also show differential

208

characteristics depending on their origin. In general the homologues > C20 with strong even-to-

209

odd C-number preference are often considered as characteristic of wax from vascular plants,

210

whereas those < C20 are believed to derive mainly from microorganisms8. In particular the α,ω-

211

alkanedioic acids and ω-hydroxyacids (C12, C14 and C16) are constituents of plant polyesters such

212

are cutin and suberin37. Other compounds as the isoprenoid ketone (6,10,14-

213

trimethylpentadecan-2-one) probably derive from phytol49. The n-alkanes (Fig. 3) had in general

214

a clear dominance of homologues > C20 (Table 3), with frequent maxima at C29 and C31, and an

215

odd/even carbon preference index (CPI) typical of epicuticular wax in vascular plants36.

216

It is often assumed that soil alkanes < C20 may have a microbial origin8. They had the highest

217

relative abundance in R2 and R4 from conifer vegetation. Alkane abundance in these samples

218

showed a trend towards a bimodal distribution (maxima at ca. C23 and C29 or C31) that could be

219

interpreted as caused by a dual origin for the homologues in the series.

220

In some distribution patterns the difference between soil pairs was small; this could indicate a

221

comparatively low environmental impact, i.e. a low intensity of the perturbation, or high soil

222

resilience, or both. Visual inspection of the histograms (Fig. 2) showed major changes in soils

223

affected by fire, which presumably produce alkane fragmentation as indicated by independent

224

studies in laboratory-controlled conditions45. The extent of this fragmentation could be reflected 9



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225

by the lack of an odd/even preference in the low molecular weight (MW) (< C20) hydrocarbons .

226

This production of a variety of short chain compounds at pyrolysis temperatures could be favored

227

by their fixation into mineral matrices, which hinder their thermo-evaporation51.

228

In any case, the proportions of alkanes and FAs did not show the same response to fire (Table

229

3), which is expected from the very different intensity of wildfires affecting soils F3 and F4 (Table

230

1).

231

The alkane patterns of the soils under deciduous forest (oak, chestnut) showed comparatively

232

small changes after clearing and cultivation, or after bush encroachment (series C or B), but

233

reforestation with pine was associated with accumulation of homologues of comparatively lower

234

Cno (Table 3).

235

In the studied soils, the classical indices based in the average chain length or the carbon

236

preference (Table 3), reflected mainly local differences between neighbor ecosystems rather

237

than systematic environmental changes. General differences were examined taking advantage of

238

additional indices such are the diversity indices of the whole alkyl series, as a possible proxy of

239

the complexity of the whole ecosystem52 or the Euclidean distances between paired control and

240

perturbed soils, to provide an objective index of the extent of the corresponding change in the

241

whole alkyl series3.

242

In fact, the extent of the changes in the alkane assemblages in samples from neighbor (relict

243

and perturbed) plots could be quantified by way of a statistical dissimilarity calculated using the

244

proportions of the different homologues in the series from the adjacent soil samples (e.g. R1 vs.

245

R2, R3 vs. R4, etc). For this purpose3, we selected the Euclidean distance ED:

246

EDi , j =

n

∑ (c − c ni

nj

)2

i =1

247

where Cn corresponds to the total abundance (normalized by the total peak area of all

248

homologues) of each compound in the series, and i and j are the different soils compared. The

249

resulting distances EDi,j which are shown in Table 3 provide a relative measure of the extent of the 10


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250


change in the whole alkyl series.

251

The interpretation of the differences between neighbor paired sites (Table 3) confirms the

252

substantial alteration produced by intense fire (F1 F2) compared with the case in which fire

253

affected more the forest canopy than the epipedon (F3 F4). On the other hand, the lowest

254

change was observed in the C1 C2 pair, which suggested as a high stability of the OM in the

255

centennial oak forest. This was also probably the case with the cleared oak forest after

256

encroachment by ameliorant bush (B1 B2) which could be interpreted as a low capacity of the

257

weak density secondary Mediterranean vegetation to introduce change in the historical period, in

258

the biogeochemical behavior of the original system31.

259

While differences between paired samples can be estimated from the Euclidean distances, the

260

complexity of the composition of a given sample could be quantitatively estimated using diversity

261

indices. The Shannon-Wiener index takes into account the number and size of subgroups in a given

262

population. It was calculated for each alkyl series using the relative abundances of the different

263

homologues; its value should increase with the complexity of the molecular population. Table 4

264

and Figs. 2 and 3 clearly show that the diversity index (H’) in the perturbed sites tended to be

265

higher than in the original ones. This trend to increased diversity was observed mainly for the

266

alkanes and, to lesser extent, FAs. This behaviour was observed in all types of perturbations,

267

including series B and C, often considered as leading to desertification or to a simplification of the

268

ecosystem structure. The increase in H’ values occurred both in situations in which the total

269

abundance of lipids in soil decreased after the perturbation (most cases) or when it increased

270

(series B, under Mediterranean bush with essential oils, adapted to xerophytic conditions).

271

There are two probable reasons for the enhanced molecular diversity after perturbation. First,

272

selectively preserved molecules from the original ecosystem would coexist with those recently

273

incorporated from the new vegetation. Other explanation could be the abiotic transformations and

274

microbial reworking of the original lipid assemblage in perturbed soils51. Since increasing molecular

275

complexity has been considered to parallel the extent to which the composition of soil lipid in 11



3

276

forest ecosystems changes as regards that of the original plant lipid , this index could inform on

277

the transformation stages of the soil system.

278

The FA patterns showed a clear even/odd preference, with C16, C22 and C24 as major members.

279

Comparison of the FAs from soils under oak forest (R1, R3) with those from soils reforested with

280

Pinus pinea and Pinus pinaster (R2, R4) showed that the former series could be differentiated by

281

maxima at C22 and C24 and a higher chain length ratio (> C20 /< C20) (Table 3). Typical C15, C17 and C19

282

iso- and anteiso-branched FAs often considered as markers of microbial activity 53 were found in

283

all samples, except B1.

284

Like the alkanes, the differences between FA series from paired plots were quantified via

285

Euclidean distances. Large transformations, in both cases accompanied by the relative

286

accumulation of low MW FAs were found after reforestation and after wildfire. There were small

287

changes in the sites cleared for cultivation or for pasture.

288

The presence of unsaturated acids was also frequent in the samples, except B1, C1 and C2, the

289

most frequent being palmitoleic (C16:1) and oleic (C18:1). In general unsaturated FAs present low

290

abundances and they could be considered as indicative of recent biogenesis, since such

291

compounds are comparatively reactive and are rapidly degraded or condensed with the active sites

292

of the soil matrix54.

293

In general terms, the information supplied by the study of FAs was similar to that suggested

294

by the alkane series. To a large extent, soil FA composition is considered to reflect the original

295

vegetation type55. All samples (Table 3) showed dominance of compounds with an even number

296

of carbons (even/odd >3) typical of a recent biogenic origin8,36. As in the case of the alkanes, for

297

FAs from series F the samples affected by fire showed a higher proportion of homologues < C20,

298

as would correspond to the thermal breakdown of higher chain homologues45,50.

299

When the impact of the disturbation was assessed in terms of the Euclidean distance between

300

FA patterns (Table 3), it is observed small changes in the case C1 C2 , which could be interpreted

301

as a substantial resilience of the relict oak forest, with a high clay content and thick humus horizon 12


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302

(10 cm). As in the case of alkanes, and when comparing plots after bush encroachment, the most

303

intense changes were produced in the soil occupied by Cistus (B4). Finally, the most marked

304

perturbation of the whole pattern of FAs corresponds, as in the case of alkanes, to the soil affected

305

by severe wildfire (F2). In these soils subjected to heating, the changes (mainly related to a

306

selective accumulation of short chain FAs, as in the case of alkanes), were proportional to the

307

intensity of the fire (F2 > F4).

308

As for the FAs, the n-alkanols (C13–C28) showed a clear odd/even preference, with a maximum at

309

C24 or C26. Besides the n-alkanols, there was a less abundant series of branched alcohols (mainly

310

C24, C26 and C28). The C14 and C16 alkanediols, frequent constituents of epicuticular lipids of

311

higher plants37 also occurred in R2. Nevertheless,the alkanol patterns did not show systematic

312

changes depending on the perturbation in the relative chain length, or in the carbon preference

313

index, but provided complementary local information on features that were not straightforwardly

314

reflected by the above alkyl compounds. In particular, the effects of bush encroachment (B2, B4)

315

could be distinguished by the increased abundance of the low MW homologues. In series R, and

316

after reforestation with pine, a dominance of the C24–C28 alkanols was observed, but in F2 and

317

F4 the chain length was shorter as a probable effect of fire.

318 319

Environmental changes reflected by the lipid signature

320

As a whole, the results suggest that the lipid molecular record in soils from Mediterranean

321

ecosystems is highly responsive to the environmental perturbations. The finding that diversity

322

indices of the alkane and FA series increase in disturbed soils vs. control soils could be

323

interpreted in terms of supposing that preexisting compounds from the original lipid signature

324

coexist with new lipids generated after the perturbation. In general, these alkyl compounds

325

were found to be an important source of environmental proxies when described via suitable

326

indices calculated from the whole homologue series.

327

Of the four pine forests, the biomarkers investigated remain efficient for plant source 13



328

reconstruction in spite of the environmental perturbations: the most stabilized ecosystems

329

showed comparatively higher proportions of dehydroabietic acid and lower of abietane- and

330

pimarane-type diterpenoids. Soils affected by forest fires showed increased proportions of

331

shorter chain alkanes and FAs as well as of natural polycyclic aromatic hydrocarbons such as

332

retene, dehydroabietin and simonellite.

333

On the other hand, some individual signature compounds were specifically found in a

334

reduced number of samples: friedooleananes were found in three soils under angiosperm

335

vegetation, whereas norambreinolid-type diterpenes occurred in soil under Cistus bush. The

336

steroids were found to prevail in a pastured site. In conclusion, the comparison of free lipid compounds in soils plots whose characteristics

337 338

have changed as a result of disturbances seems to indicate as if there were a "memory of soil

339

lipids" that revealed environmental impact in soil. In all cases, the signature of lipid assemblages

340

reflected changes in land use with alkyl homologous series being especially responsive to the

341

impact of fires. From the chemometric viewpoint, the environmental impact was associated with

342

enhanced diversity of alkane homologues, and Euclidean distance between alkane abundances

343

in paired control and disturbed sites acts as a proxy for the extent of soil disturbation.

344 345

ACKNOWLEDGMENTS

346

Financial support by Spanish MINECO project CGL2013-43845-P is acknowledged. The authors

347

would like to express their sincere appreciation to Dr. J.R. Maxwell by his helpful comments and

348

suggestions to a previous version of the document.

349 350

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351

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soil organic matter by spectroscopic and thermal degradation methods. In: Fire Effects in

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498

20


Page 22 of 34

Page 23 of 34

499


Figure captions

500 501

Fig. 1. TIC trace of methylated soil lipids from Quercus ilex subsp. ballota (R1), Pinus pinea (R2),

502

Pinus halepensis (F1) and Pinus halepensis affected by forest fire (F2). FA, fatty acids; AL, alkanes;

503

OH, alkanols; numbers on the peaks refer to Table 4.

504 505

Fig. 2. Monoterpenes, diterpenes and sesquiterpenes (as methyl esters), triterpenoids and

506

sterols identified in soil lipids.

507 508

Fig. 3. Distribution patterns (Cno vs. relative abundance) of selected series of alkyl compounds

509

(for information on the remaining series see Table 3). The n-alkanes indicate change as results of

510

wildfires (F1F2 and F3F4). The FAs indicate changes due to clearing and cultivation [oak

511

forest to cereal fields (C1C2) or ash forest to grazing pasture (C3C4)]. The alkanol series

512

reflect changes attributable to bush encroachment by species of Fabaceae (broom) or Cistaceae

513

(B2 and B4 respectively). Sample labels refer to Table 1. H’, diversity index; EDS1,S2, Euclidean

514

distance between S1 and S2, calculated to quantify the extent of the change after perturbation;

515

the total abundances (normalized to the total peak area of all homologues) of alkanes or FAs were

516

used as descriptors.

517 518 519

21



Page 24 of 34

Table 1 Soil classification and general analytical characteristics of samples soils in Central Spain Ref. Ecosystem R1 R2 R3 R4

Relict evergreen oak forest Reforested pine forest Relict evergreen oak forest Reforested pine forest

C1

Relict oak forest

C2

Cultivated wheat field

C3

Relict ash forest

C4

Pastured site

B1

Relict chesnut forest

B2

Secondary bush

B3

Control evergreen oak forest

B4

secondary bush

F1

Pine forest

F2

Burned pine forest (high intensity fire)

F3

Pine forest

F4

Burned pine forest (mainly affecting forest canopy)

Soil type (IUSS Working Group WRB, 2015) Eutric Cambisol (Loamic, Humic ) Eutric Cambisol (Loamic, Humic) Cambic Folic Phaeozem (Loamic, Humic) Eutric Cambisol (Loamic, Humic) Eutric Folic Cambisol (Loamic, Humic) (Rendzic Leptosol (Loamic) Eutric Cambisol (Loamic, Humic) Eutric Cambisol (Loamic, Humic) Eutric Folic Cambisol (Loamic, Humic) Dystric Cambisol (Loamic, Humic) Eutric Cambisol (Loamic, Humic Eutric Cambisol (Loamic, Humic Calcaric Cambisol (Loamic, Humic) Calcaric Cambisol (Loamic, Humic) Cambic Umbrisol (Loamic, Hyperdystric Hyperdystric Cambisol (Loamic, Humic)

Universal Transverse Parent rock Vegetation Mercator (UTM) Quercus ilex subsp. ballota in addition to Rosmarinus officinalis, Daphne gnidium, 4488–411 Granite Cistus ladanifer Pinus pinea in addition to Cistus ladanifer 4488–411 Granite 4490–403

Granite

Quercus pyrenaica, in addition to Prunus spinosa, Rosa canina

4490–403

Granite

Pinus pinaster, in addition to Cistus ladanifer

4472–476

Limestone

Quercus ilex subsp. ballota

4472–476

Limestone

Triticum aestivum

4526–451

Granite

Fraxinus angustifolia, in addition to Rosa canina, Paeonia coriacea

4526–451

Granite

Poa bulbosa, Trifolium dubium, Trifolium campestre in addition to Fraxinus angustifolia, Rosa canina, Paeonia coriacea and Micropyrum tenellum

4465–371

Granite

Castanea sativa, Genista sp., Rosa canina, Vicia sp. and Poaceae

4465–371

Granite

Cytisus scoparius, Genista sp., Retama sphaerocarpa and Lavandula stoechas

4493–401

Granite

Quercus ilex subsp. ballota, Cistus ladanifer, Rosmarinus officinalis and Daphne gnidium

4493–401

Granite

Cistus ladanifer, Daphne gnidium, Retama sphaerocarpa, and residual forest of Quercus ilex subsp. ballota,.

4459–450

Limestone

Pinus halepensis, Eryngium campestre and Reseda sp.

4459–450

Limestone

Pinus halepensis, Eryngium campestre and Reseda sp.

4553–52

Gneiss

Pinus sylvestris, Erica sp., Pteridium aquilinum and Cytisus scoparius

4553–452

Gneiss

Pinus sylvestris, Erica sp., Pteridium aquilinum and Cytisus scoparius

22


Page 25 of 34


Table 2

Analytical characteristics of soil samples (0–10 cm depth)

Sample

Altitude

Slope

pH

Clay

m asl

(%)

(H2O)

g kg

Ca

Textural type (USDA)

-1

g kg

Lipidb

C/N -1

CECc

-1

g kg

Sd

cmolc kg

-1

-1

cmolc kg

R1

880

5

7.5

101

Sandy loam

170

16.5

1.45

47.1

33.9

R2

855

0

6.0

30

Loamy sand

67

24.7

0.59

12.2

7.6

R3

1150

15

5.9

158

Sandy loam

39

11.3

0.17

17.2

10.4

R4

1240

15

6.2

55

Sandy loam

65

29.6

0.54

19.0

13.3

C1

870

0

7.9

27

Silt loam

95

15.3

0.29

41.2

41.2

C2

870

0

8.4

228

Silt loam

17

12.7

0.10

21.2

21.2

C3

950

2

6.8

113

Sandy loam

92

13.3

0.49

21.8

20.8

C4

950

2

6.0

126

Sandy loam

49

13.1

0.18

12.6

10.2

B1

840

20

6.2

134

Loam

61

15.5

0.08

17.1

11.1

B2

825

5

6.2

45

Loamy sand

57

13.0

0.13

10.6

4.7

B3

1015

15

7.1

51

Sandy loam

44

14.2

0.13

15.0

7.5

B4

990

20

6.5

86

Sandy loam

88

17.1

0.37

25.0

16.8

F1

630

0

6.9

120

Silt loam

213

14.5

9.50

70.6

49.7

F2

624

8

8.7

125

Sandy loam

39

12.3

0.05

13.5

13.5

F3

1580

20

4.6

77

Sandy loam

69

14.7

0.42

23.5

2.1

1615

15

5.7

64

Sandy loam

64

8.2

0.14

41.2

4.0

0.1

30

5

1.4

1.8

5.11

F4 LSD a

e b

c

d

0.02 +

+

2+

2+

e

Total oxidizable soil C; Soxhlet extraction with petroleum ether; cation exchange capacity; sum of exchangeable bases (Na + K + Ca + Mg ); least significant difference based

on adjacent spatial replicates.

23



Page 26 of 34

Table 3 Relative concentrationa, ratios and statistical indices calculated from alkanes, FAs and alkanols in the soils Compound type, ratios

R1

R2

Total alkanes

27.0

R3

3.0

R4

22.2

3.1

B3

B4

68.1

49.6

45.9

22.4

4

283

197

509

202

C2

C3

C4

B1

47.3

33.8

38.8

21.9

Linear/branched

187

2n+1/2n

B2

C1

F1

9

2

4

4

7

6

12

4

6

8

9

8

> C20/C20/

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