Marlène Lavrieux

Jérémy Jacob

Claude Le Milbeau

Renata Zocatelli

Kazuo Masuda

Jean-Gabriel Bréhéret

Jean-Robert Disnar

Jean-Gabriel Bréheret

Occurrence of triterpenyl acetates in soil and their potential as chemotaxonomical markers of Asteraceae

Keywords: Asteraceae, soil, biomarkers, pentacyclic triterpenyl acetates

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Understanding ecosystem response under natural (climatic) and anthropogenic constraints is of crucial importance for anticipating the consequences of current global change [START_REF] Dearing | Climate-human-environment interactions: resolving our past[END_REF]. Land use changes have direct consequences for the budget of greenhouse gases at the global scale. At a more local scale, it has been demonstrated that soil properties result from a long history of land use [START_REF] Hurtt | The underpinnings of land use history: three centuries of global gridded land use transitions, wood-harvest activity, and resulting secondary lands[END_REF]. The reconstruction of recent land use change can be approached through historical archives.

For example, the impact of drastic land use change on biodiversity is particularly well documented for the middle mountains of the French Massif Central during the twentieth century [START_REF] Chassagne | Inventaire Analytique de la Flore d'Auvergne et Contrées Limitrophes des Départements Voisins[END_REF][START_REF] Antonetti | Atlas de la Flore d'Auvergne[END_REF].

With respect to more ancient times, the scarcity of reliable written sources necessitates an investigation of natural archives (lake sediments, peat and speleothems) in order to unravel past natural and anthropogenic impacts on environments. These environmental records retain information about such interactions in a well constrained time frame. Deciphering such interactions involves reconstruction of ancient land use via multidisciplinary studies (e.g. palaeoecology, archaeology, sedimentology; e.g. [START_REF] Berglund | The cultural landscape during 6000 years in southern Sweden: the Ystad Project[END_REF] that provide different and complementary information.

While the potential of sediments as natural archives has largely been demonstrated, soil, although a fundamental substrate ofor human subsistence and direct support of agricultural practices, has not received comparable interest. Soil is known to be disturbed in terms of physical properties, organic matter (OM) content and thereby of fertility when affected by land use change [START_REF] Rumpel | Impact of land use change on the molecular composition of soil organic matter[END_REF]. The after effects of such changes remain perceptible in terms of biodiversity, and of chemical and structural soil properties, at least at the historical scale [START_REF] Dupouey | Irreversible impact of past land use on forest soil and biodiversity[END_REF]. However, only a few studies have focussed on the direct recording of land use at the molecular level (e.g. [START_REF] Van Bergen | Organic geochemical studies of soils from the Rothamsted Classical Experiments--l. Total lipid extracts, solvent insoluble residues and humic acids from Broadbalk Wilderness[END_REF][START_REF] Bull | Organic geochemical studies of soils from the Rothamsted Classical Experiments -II, Soils from the Hoosfield Spring Barley Experiment treated with different quantities of manure[END_REF]. These studies have shown that the lipid content is directly linked to the current overlying vegetation, since the major part of the soil lipids originates from plants and is well preserved (e.g. [START_REF] Dinel | Soil lipids: origin, nature, content, decomposition, and effect on soil physical properties[END_REF][START_REF] Van Bergen | Organic geochemical studies of soils from the Rothamsted Classical Experiments--l. Total lipid extracts, solvent insoluble residues and humic acids from Broadbalk Wilderness[END_REF][START_REF] Bull | Organic geochemical studies of soils from the Rothamsted Classical Experiments -II, Soils from the Hoosfield Spring Barley Experiment treated with different quantities of manure[END_REF]. This makes them a reliable tool as molecular biomarkers, i.e. as compounds preserved intact or slightly modified in natural archives (and aerosols), the structure of which allows unequivocal identification of their biological source (e.g. [START_REF] Van Bergen | Organic geochemical studies of soils from the Rothamsted Classical Experiments--l. Total lipid extracts, solvent insoluble residues and humic acids from Broadbalk Wilderness[END_REF]Oros and Simoneit, 2001a,b;[START_REF] Simoneit | Molecular indicators (biomarkers) of past life[END_REF][START_REF] Simoneit | A review of current applications of mass spectrometry for biomarker / molecular tracer elucidations[END_REF][START_REF] Killops | An Introduction to Organic Geochemistry[END_REF]. Such molecular markers benefit from increasing interest for studies dealing with vegetation change or human activity (e.g. [START_REF] Hjulström | Identification of activity area signatures in a reconstructed Iron Age house by combining element and lipid analyses of sediments[END_REF]. Amongst these, pentacyclic triterpenes are of special interest for organic geochemists exploring the distributions of lipids in soil, sediments and peat as remnants of past vegetation. For example, those with a lupane, oleanane or ursane structure, as well as their diagenetic derivatives, in soil and sediments are classically used as tracers of angiosperms [START_REF] Cranwell | Organic geochemistry of lacustrine sediments: triterpenoids of higher plant origin reflecting post-glacial vegetational succession[END_REF][START_REF] Jacob | Contrasted distributions of triterpene derivatives in the sediments of Lake Caçó reflect paleoenvironmental changes during the last 20,000 yrs in NE Brazil[END_REF]. However, as a result of their wide structural (skeletal) diversity (oleanane, ursane, lupane, taraxerane…), potential isomerism and associated functional groups, several recent studies have shown that, in favourable cases, pentacyclic triterpenes can be discriminant for more constrained taxa.

For example, Gramineae are the most common producers of pentacyclic triterpenes bearing a MeO group at C-3 [START_REF] Ohmoto | Triterpenoids of the Gramineae[END_REF][START_REF] Jacob | Pentacyclic triterpene methyl ethers in recent lacustrine sediments (Lagoa do Caçó, Brazil)[END_REF][START_REF] Zocatelli | Molecular evidence for recent turf cultivation in Northeast Brazil (Lagoa do Boqueirão, RN State)[END_REF]. In particular, more specific relationships can be established, such as that allowing the linking of miliacin (olean-18-en-3-ol methyl ether) in sediments from Lake le Bourget (French Alps) to Panicum miliaceum (broomcorn millet) cultivated in the watershed [START_REF] Jacob | Millet cultivation history in the French Alps as evidenced by a sedimentary molecule[END_REF][START_REF] Jacob | Impacts of new agricultural practices on soil erosion during the Bronze Age in French Prealps[END_REF].

Here we report the presence of a series of pentacyclic triterpenyl acetates in a soil developed under pasture in the catchment of Lake Aydat (Massif Central, France).

Following description of the different compounds analysed by way of gas chromatography-mass spectrometry (GC-MS), we discuss their potential biological sources and finally evaluate their potential for palaeoenvironmental studies.

Setting, materials and methods

Location and general context

Lake Aydat is ca. 25 km SW of Clermont-Ferrand, in the volcanic French Massif Central. The small catchment of the lake (ca. 30 km²) is at an altitude ranging from 825 m (lake level) to ca. 1,300 m above sea level (a.s.l.). The tops of the volcanoes, the highest points, are covered by coniferous forest (mainly of Picea sp.), whereas the slopes are covered by pasture/grassland and, to a lesser extent, shrubs. The geology, relating mainly to a volcanic origin, led to the development of andisoils as a result of rapid chemical erosion in the presence of OM [START_REF] Jones | Soil Atlas of Europe[END_REF]. Typically, a homogeneous organo-mineral sandy-silty horizon A developed above the C horizon.

These slightly acidic soils are well drained and quite shallow (< 30 cm).

Materials and methods

A total of 35 soil samples were taken from the lake catchment in the autumn of 2008. They represent the diversity of land use, as described above, and were collected at different altitude and orientation, so that each condition prevailing in the catchment was taken into account. In the absence of recognizable layers, soils were sub-sampled in slices 2 cm thick. The 0-2 and 2-4 cm depth slices were analysed in random order, since they were considered the most representative of the current vegetation cover.

Samples were dried at 40 °C for 48 h in an oven, crushed in a mortar and sieved at 2 mm. An aliquot (ca. 2 g) of each was extracted by way of automatic solvent extraction with a Dionex® ASE 200 using CH2Cl2:MeOH (1:1). After removal of the solvent under N2, the extract was separated into neutral, acidic and polar fractions on aminopropyl-bonded silica as described by [START_REF] Jacob | Pentacyclic triterpene methyl ethers in recent lacustrine sediments (Lagoa do Caçó, Brazil)[END_REF]. The neutral fraction was further separated into aliphatics, aromatics, ethers and esters, ketones and acetates, and alcohols by way of flash chromatography with a Pasteur pipette filled with activated silica (24 h at 120 °C, then deactivated with 5% H2O) and using a sequence of solvents of increasing polarity. The fraction containing triterpenyl acetates and ketones was eluted with a mixture of hexane:EtOAc (9:1) and 5-cholestane was added prior to analysis via GC-MS with a TRACE-GCQ Polaris. The GC instrument was fitted with a Rtx-5MS column (30 m x 0.25 mm i.d., 0.25 µm film thickness; 5m column guard). The operating conditions were: 40 °C (1 min) to 120 °C at 30 °C min -1 , then from 120° to 300 °C (held 30 min) at 5 °C min -1 . Samples were injected in splitless mode, with the injector at 280 °C. He was the carrier gas at a constant flow of 1 ml min -1 . The mass spectrometer was operated in the electron ionization (EI) mode at 70 eV and scanned from m/z 50 to 600. Component identification was based on comparison of retention times and mass spectra with authentic standards. The concentration of triterpenyl acetates was estimated by measuring peak areas in ion chromatograms. After calculating a correction factor between the peak area on the specific ion chromatogram and the peak area on the total ion current (TIC) chromatogram, the TIC area of triterpenyl acetates was measured relative to that of the standard (5α-cholestane) and to the mass of sample extracted. The identification of all compounds, except isopichierenyl acetate, was confirmed using authentic standards.

Results

This study reports on the triterpenyl acetates in a single soil sample that showed the widest diversity among the 35 samples. Variation in the distributions, probably reflecting various changes in biological input resulting from local conditions in the catchment, are not discussed here.

Fifteen triterpenyl acetates eluted in the 44-52 min tR range under our GC conditions (Fig. 1). The structures discussed are displayed together with the mass spectra in Fig. 2.

The spectra of all triterpenyl acetates display ions at m/z 468, 453, 393 408

([M+], [M + -15], [M + -60],
i.e. loss of the acetate group, and [M + -60-15], respectively), with an intensity depending on the compound. Compounds 1, 2, 3 and 6 elute in the 44-47 min tR range and are characterised by intense m/z 189, 203, 204 and 218 fragments attesting to a taraxerane, oleanane or ursane structure [START_REF] Shiojima | Mass spectra of pentacyclic triterpenoids[END_REF].

Considering the relative retention times, 2, 3 and 6 were assigned as olean-13(18)-en-3-yl acetate (-amyrin acetate), olean-12-en-3-yl acetate (-amyrin acetate) and urs-12-en-3-yl acetate (-amyrin acetate) respectively, whereas the rather intense fragments at m/z 269, 329 and 344 in the spectrum of compound 1 attest to a tarax-14en-3-yl acetate (taraxeryl acetate) structure.

The spectra of 10 and 11 are also characterized by intense fragments at m/z 189 and 204. Their late elution time vs. 2 and 3, combined with the presence of an ion at m/z 249, allowed identification of 10 as taraxast-20-en-3-yl acetate (-taraxasteryl acetate) and 11 as taraxast-20(30)-en-3-yl acetate (taraxasteryl acetate). This assignment was confirmed by comparison with authentic standards.

Intense fragments at m/z 259 and 274 in the spectrum of 5 are diagnostic for D:B-friedo-oleanane structures [START_REF] Shiojima | Mass spectra of pentacyclic triterpenoids[END_REF], consistent with glutin-5-en-3-yl acetate (glutinyl acetate).

The spectra of 4, 8, 9, 12, 13 and 15 are characterized by intense ion at m/z 229, 289, 241 and 301, with a minor contribution from ions at m/z 255 and 393 (Fig. 2). These features are characteristic for D:C-friedo-ursen-3-ol and D:C-friedo-oleanen-3-ol acetates (bauerenyl and multiflorenyl acetates, respectively) and are also found in swertenyl acetates [START_REF] Shiojima | Mass spectra of pentacyclic triterpenoids[END_REF]. The use of authentic standards and relative retention times allowed assignment of 4 as bauer-8-en-3-yl acetate (isobauerenyl acetate), 9 as bauer-7-en-3-ol acetate (bauerenyl acetate), 8 as multiflor-7-en-3-yl acetate (multiflorenyl acetate), 12 as pichier-8-en-3-yl acetate (isopichierenyl acetateno standard available), 13 as pichier-9(11)-en-3-yl acetate (pichierenyl acetate) and 15 as pichier-7-en-3-yl acetate (swertenyl acetate). Finally, 7 was assigned as lup-20(29)en-3-yl acetate (lupeyl acetate) and 14 as gammacer-16-en-3-yl acetate (gammacerenyl acetate) on the basis of their mass spectra [START_REF] Shiojima | Mass spectra of pentacyclic triterpenoids[END_REF] and authentic standards.

Taraxeryl acetate (1.10 µg/g soil), pichierenyl acetate (0.91 µg/g soil), glutinyl acetate (0.81 µg/g soil), taraxasteryl acetate (0.29 µg/g soil), -taraxasteryl acetate (0.07 µg/g soil) and isopichierenyl acetate (0.54 µg/g soil) were the most abundant compounds in the sample. In all the other samples, the concentration rarely reach 1 µg/g soil. For comparison, the concentrations in plants range from a few tens of µg to a few mg per gram dried plant, with a strong variability as to whether roots or aerial parts are considered (i.e. [START_REF] Bohlmann | A guaianolide and other constituents from Lychnophora species[END_REF]Shiojima et al., 1989b;[START_REF] Lu | Terpenes from Liatris ohlingerae[END_REF][START_REF] Tsao | New diterpenoids and the bioactivity of Erythrophleum fordii[END_REF].

Discussion

Distribution of triterpenyl acetates in plants

Our literature survey of triterpenyl acetates sources led to an inventory of > 460 plants known to produce these compounds. Most information concerning Asteraceae (432 species among the 460) was extracted from the "Bohlmann Files" database (Jakupovic et al., 2011), which concerns all the natural components of this family. This information was completed with other data on the distribution of triterpenyl acetates in Asteraceae. Additional data on the occurrence of the compounds in other plant taxa allowed testing of the chemotaxonomical value of the different triterpenyl acetates.

Depending on bibliographical source, the degree of detail is highly variable for the different taxa. The resulting database evidently suffers from bias linked to a more specific interest toward a given plant taxa or a given chemical family. Nevertheless, the inventory provides key information on the distribution of pentacyclic triterpenyl acetates in plants.

The most commonly reported triterpenyl acetates are lupeyl acetates, comprising lup-20(29)-en-3-yl acetate, lup-19(20)-enyl acetate, tarolupenyl acetate [lup-19(21)en-3-yl acetate], epilupeyl acetate [epi-lup-20(29)-en-3-yl acetate], lactucenyl acetate (D-friedo-lup-14-en-3-yl acetate; [START_REF] Shinozaki | Composite constituent: lactucenyl acetate, a novel migrated lupane triterpenoid from Lactuca indica L. Revision of structure of tarolupenyl acetate[END_REF] and neolupenyl acetate (lup-12-en-3-yl acetate), the first being by far the most reported. Of the Asteraceae, 416 species are reputed to contain lupeyl acetates (Jakupovic et al., 2011;[START_REF] Madrigal | Carduus nigrescens seed oila rich source of pentacyclic triterpenoids[END_REF], but this component is also present in various other families such as Amaranthaceae, Cucurbitaceae, Euphorbiaceae, Moraceae and Apocynaceae (e.g. Jewers and Manchada, 1970;[START_REF] Dutta | Taraxasterol and its derivatives from Cirsium arvense[END_REF][START_REF] Woldu | Styrenes from Dorstenia barnimiana[END_REF][START_REF] Yoshizumi | Medicinal foodstuffs. XI. Histamine release inhibitors from wax gourd, the fruits of Benincasa hispida[END_REF][START_REF] Medeiros | A new natural quaternary indole alkaloid isolated from Tabernaemontana laeta Mart. (Apocynaceae)[END_REF][START_REF] Gasparetto | Extracts from Alternanthera maritima as natural photosensitizers in photodynamic antimicrobial chemotherapy (PACT)[END_REF]. Taraxasteryl and taraxasteryl acetates were overwhelmingly detected in Asteraceae, with 153 and 29 occurrences, respectively [START_REF] Dutta | Taraxasterol and its derivatives from Cirsium arvense[END_REF][START_REF] Madrigal | Carduus nigrescens seed oila rich source of pentacyclic triterpenoids[END_REF][START_REF] Jordon-Thaden | Chemistry of Cirsium and Carduus: a role in ecological risk assessment for biological control of weeds?[END_REF]Jakupovic et al., 2011) and also in a few Apocynaceae (6 and 2 occurrences, respectively; Jewers and Manchada, 1970;[START_REF] Thakur | Latex extractables of Calotropis gigantea[END_REF][START_REF] Abbott | Major extractable components in Asclepias linaria (Asclepiadaceae) and Ilex verticillata (Aquifoliaceae), two potential hydrocarbons crops[END_REF][START_REF] Sen | Flavonol glycosides from Calotropis gigantea[END_REF][START_REF] Medeiros | A new natural quaternary indole alkaloid isolated from Tabernaemontana laeta Mart. (Apocynaceae)[END_REF][START_REF] Zhang | Studies on chemical constituents in root tuber of Cynanchum auriculatum[END_REF][START_REF] Yin | Chemical constituents in the root of Cynanchum auriculatum[END_REF][START_REF] Pereira | Chemical constituents from Tabernaemontana catharinensis root bark: a brief NMR review of indole alkaloids and in vitro cytotoxicity[END_REF]. Bauerenyl acetate was found in three Asteraceae spp., four Apocynaceae and Euphorbia chrysocoma [START_REF] Cava | Triterpene constituents of Tabernaemontana laurifolia and Haplophyton cimicidum[END_REF][START_REF] Cicció-Alberti | Algunos constituyentes de los frutos y de las hojas de Tabernaemontana longipes Donn. Smith[END_REF][START_REF] Lao | Studies on the constituents of Artemisia rubripes Nakai[END_REF][START_REF] Shi | A new bisabolane sesquiterpenoid from Euphorbia chrysocoma[END_REF][START_REF] Pereira | Chemical constituents from Tabernaemontana catharinensis root bark: a brief NMR review of indole alkaloids and in vitro cytotoxicity[END_REF]Jakupovic et al., 2011).

Isobauerenyl acetate was only found in Euphorbia fischeriana [START_REF] Liu | Studies on chemical constituents from Euphorbia fischeriana Steud[END_REF] and in two Asteraceae spp.: Picris hieracioides (Shiojima et al., 1989a) and Centaurea aspera [START_REF] Picher | Components of hexane extracts of the flowers of Centaurea aspera L. ssp. stenophylla (Dufour) Nyman[END_REF]; -amyrin acetate occurs in three Asteraceae spp.: Vernonia cinerea [START_REF] Misra | Chemical constituents of Vernonia cinerea, Part I. Isolation and spectral studies of triterpenes[END_REF], Inula britannica and Echinops echinatus (Jakupovic et al., 2011) and in the Phyllanthaceae sp., Phyllanthus polyanthus [START_REF] Ndlebe | Triterpenoids from the African tree Phyllanthus polyanthus[END_REF].

The biosynthesis of lupeyl, taraxasteryl, -taraxasteryl, bauerenyl, as well as that of isobauerenyl acetates therefore appears almost exclusively constrained to Asteraceae. The lack of data concerning -amyrin acetate does not allow discussion of its specificity. Remarkable is the detection of pichierenyl, isopichierenyl, gammacer-16en-3-yl and swertenyl acetates in our soil samples since these compounds have only been described in the roots of the hawkweed oxtongue Picris hieracioides (Shiojima et al., 1989a,b;[START_REF] Kataria | Phytochemical investigation of medicinal plant Cnicus wallichii and Cnicus benedictus L[END_REF].

Although taraxeryl and -amyrin acetates are also found in Asteraceae (9 and 29 spp., respectively; [START_REF] Yang | Chemical constituents of Artemisia dalailamae Kraschen[END_REF][START_REF] Madrigal | Carduus nigrescens seed oila rich source of pentacyclic triterpenoids[END_REF]Jakupovic et al., 2011), they were also detected in various taxa such as Apocynaceae, Aceraceae, Euphorbiaceae, Crassulaceae, Betulaceae and Moraceae (e.g. [START_REF] Dutta | Taraxasterol and its derivatives from Cirsium arvense[END_REF]Stevens et al., 1994a;[START_REF] Anjaneyulu | Chemical investigation of some Indian plants[END_REF][START_REF] Van Bergen | Organic geochemical studies of soils from the Rothamsted Classical Experiments--l. Total lipid extracts, solvent insoluble residues and humic acids from Broadbalk Wilderness[END_REF][START_REF] Vilegas | Further triterpenes, steroids and furocoumarins from Brazilian medicinal plants of Dorstenia genera (Moraceae)[END_REF][START_REF] Wada | Three new diarylheptanoid glycosides from Alnus japonica[END_REF][START_REF] Pereira | Chemical constituents from Tabernaemontana catharinensis root bark: a brief NMR review of indole alkaloids and in vitro cytotoxicity[END_REF]. Multiflorenyl acetate was found in Cirsium sp. and Ixeris chinensis (Asteracae; [START_REF] Ulubelen | Triterpenic and steroidal compounds of Cnicus Benedictus[END_REF][START_REF] Shiojima | Composite constituent: novel triterpenoid, 17-epi-lupenyl acetate, from aerial parts of Ixeris chinensis[END_REF][START_REF] Kataria | Phytochemical investigation of medicinal plant Cnicus wallichii and Cnicus benedictus L[END_REF]Jakupovic et al., 2011), as well as in Tabernaemontana longipes (Apocynaceae; Cicció-Alberti and Hoet, 1981), Euphorbia guyoniana (Euphorbiaceae; [START_REF] Haba | Diterpenoids and triterpenoids from Euphorbia guyoniana[END_REF], Sedum brevifolium and S. meyeri-johannis (Stevens et al., 1994a), and in Polypodium niponicum (Polypodiaceae; [START_REF] Ageta | Fern constituents: pentacyclic triterpenoids isolated from Polypodium niponicum and P. formosanum[END_REF]. -Amyrin acetate was described in 11 Asteraceae spp. (Jakupovic et al., 2011), but also in various families such as Apocynaceae spp. [e.g. Tabernaemontana laeta [START_REF] Medeiros | A new natural quaternary indole alkaloid isolated from Tabernaemontana laeta Mart. (Apocynaceae)[END_REF], Crassulaceae (Stevens et al., 1994a), Sapotaceae (e.g. Pouteria tomentosa; [START_REF] Anjaneyulu | Chemical investigation of some Indian plants[END_REF], Moraceae (e.g. Antiaris africana; [START_REF] Okogun | Triterpenoids and betaines from the latex and bark of Antiaris Africana[END_REF], or Balanophoraceae (Balanophora abbreviate; [START_REF] Yadagiri | Triterpenoids from Balanophora abbreviata and Balanophora indica[END_REF]]. Therefore, taraxeryl, -amyrin, amyrin and multiflorenyl acetates cannot be considered as being specific of Asteraceae.

Finally, glutinyl acetate has not been reported in Asteraceae but has been in Kalanchoe daigremontiana (Crassulaceae;[START_REF] Van Maarseveen | Composition of the epicuticular and intracuticular wax layers on Kalanchoe daigremontiana (Hamet et Perr. De la Bathie) leaves[END_REF], Acer mandschuricum (Aceraceae; [START_REF] Ding | Triterpene compounds isolated from Acer mandshuricum and their anti-inflammatory activity[END_REF], Erythrophleum fordii (Fabaceae; [START_REF] Tsao | New diterpenoids and the bioactivity of Erythrophleum fordii[END_REF], as well as in Dorstenia sp. and Maquira coriacea (Moraceae; [START_REF] Woldu | Styrenes from Dorstenia barnimiana[END_REF][START_REF] Mitaine-Offer | Constituents of the trunk bark of Maquira coriacea[END_REF].

Comparison of soil triterpenyl acetate diversity with local plant sources

According to the survey by [START_REF] Antonetti | Atlas de la Flore d'Auvergne[END_REF], 97 species of Asteraceae occur in the catchment of Lake Aydat, ten being reputed to produce triterpenyl acetates (Table 2): the greater burdock (Arctium lappa), the musk thistle (Carduus nutans), the creeping thistle (Cirsium arvense), the mouse-ear hawkweed (Hieracium pilosella), the cotton thistle (Onopordum acanthium, the bristly oxtongue (Picris echioides), the hawkweed oxtongue (Picris hieracioides), the goldenrod (Solidago virgaurea), the common sowthistle (Sonchus oleraceus) and the dandelion (Taraxacum sp.).

Picris hieracioides, largely present in the area, appears as the most evident primary source of triterpenyl acetates in our samples because it is the only plant reputed to produce isopichierenyl, pichierenyl, swertenyl and gammacer-16-en-3-yl acetates (Shiojima et al., 1989a;[START_REF] Anjaneyulu | Chemical investigation of some Indian plants[END_REF]. Because it also produces -amyrin, -amyrin, lupeyl, bauerenyl, isobauerenyl, taraxasteryl and -taraxasteryl acetates (Shiojima et al., 1989a;[START_REF] Anjaneyulu | Chemical investigation of some Indian plants[END_REF][START_REF] Kataria | Phytochemical investigation of medicinal plant Cnicus wallichii and Cnicus benedictus L[END_REF], it could also have contributed to these compounds in soil.

Other local sources of -amyrin acetate from Asteraceae can alternatively be provided by Cirsium arvense [START_REF] Tulloch | Epicuticular wax of Cirsium arvense[END_REF] and Solidago virgaurea [START_REF] Choi | Phytochemical constituents of the aerial parts from Solidago virga-aurea var. gigantean[END_REF]. Similarly, taraxasteryl acetate can be produced by up to seven species of Asteraceae encountered in the catchment (Arctium lappa, Carduus nutans, Cirsium arvense, Onopordum acanthium, Sonchus oleraceus and Taraxacum officinale; Jakupovic et al., 2011;[START_REF] Jordon-Thaden | Chemistry of Cirsium and Carduus: a role in ecological risk assessment for biological control of weeds?[END_REF][START_REF] Dutta | Taraxasterol and its derivatives from Cirsium arvense[END_REF][START_REF] Khalilova | Isolation and crystal structure of taraxasteryl acetate from Onopordum acanthium[END_REF][START_REF] Hänsel | Sesquiterpenlacton--D-glucopyranoside sowie ein neues eudesmanolid ais Taraxacum officinale[END_REF].  -Taraxasteryl acetate is also synthesised by Cirsium arvense [START_REF] Tulloch | Epicuticular wax of Cirsium arvense[END_REF], whereas lupeyl acetates are found in Cirsium arvense, Hieracium pilosella, Picris echioides and Sonchus oleraceus [START_REF] Tulloch | Epicuticular wax of Cirsium arvense[END_REF]Jakupovic et al., 2011).

Most of the triterpenyl acetate distribution in our soil sample can thus be explained by Asteraceae species found in the surrounding vegetation, except for taraxeryl, glutinyl, multiflorenyl and -amyrin acetates (Table 2). Because current knowledge of the distribution of these compounds does not allow explanation of their presence in our sample, alternative explanations can be invoked, each exemplified by one or two compounds: (i) Taraxeryl, glutinyl, -amyrin and multiflorenyl acetates may be produced by other Asteraceae spp. that occur in the catchment but have not yet benefitted from a complete phytochemical survey. Due to their belonging to genera in which other species are known to synthesize triterpenyl acetates, they are likely to do so as well (Table 2). In addition to Cirsium arvense, six Cirsium spp. have been described in the Lake Aydat catchment (C. acaule, C. dissectum, C. eriophorum, C. erisithales, C. palustre and C. vulgare;[START_REF] Antonetti | Atlas de la Flore d'Auvergne[END_REF], but whose pentacyclic triterpenyl acetate content has not been investigated. Because multiflorenyl acetate was described in Cirsium sp. and C. benedictus (Asteraceae; [START_REF] Ulubelen | Triterpenic and steroidal compounds of Cnicus Benedictus[END_REF][START_REF] Kataria | Phytochemical investigation of medicinal plant Cnicus wallichii and Cnicus benedictus L[END_REF], it is tempting to suppose that one of the Cirsium spp. in the catchment could also produce multiflorenyl acetate. Similarly, because taraxeryl acetate was described in Artemisia dalailamae [START_REF] Yang | Chemical constituents of Artemisia dalailamae Kraschen[END_REF], it could possibly originate from Artemisia verlotiorum or A. vulgaris (Asteraceae), both reported in the area. Likewise, -amyrin acetate was described in Inula britannica, while another species of the genera, I. conyza, grows in the area and could therefore be the source of this compound in the soil sample. According to phytochemical data, this compound is only described in Erythrophleum fordii (Fabaceae: [START_REF] Tsao | New diterpenoids and the bioactivity of Erythrophleum fordii[END_REF], Kalanchoe daigremontiana (Crassulaceae: van [START_REF] Van Maarseveen | Composition of the epicuticular and intracuticular wax layers on Kalanchoe daigremontiana (Hamet et Perr. De la Bathie) leaves[END_REF], Acer mandshuricum (Aceraceae: [START_REF] Ding | Triterpene compounds isolated from Acer mandshuricum and their anti-inflammatory activity[END_REF], Dorstenia sp. and Maquira coriacea (Moraceae: [START_REF] Woldu | Styrenes from Dorstenia barnimiana[END_REF][START_REF] Mitaine-Offer | Constituents of the trunk bark of Maquira coriacea[END_REF], none of these species or genus being found in the catchment except for Acer sp. (A. pseudoplatanus, A. campestre subsp. campestre and A. platanoides;[START_REF] Antonetti | Atlas de la Flore d'Auvergne[END_REF]. In the area, the occurrence of Acer species could thus explain the presence of glutinyl acetate in the soil sample.

(iii) The compounds are produced by one or several of the already identified potential sources of triterpenyl acetates. Asteraceae spp. developed around Lake Aydat could synthesize distinct triterpenyl acetates distinct from those in the same species cited in the literature. Such a variability in chemodemes (i.e. biochemical races -plants from the same species producing distinct chemicals depending on location, environmental conditions or slight genetic differences) have, for example, been described for pentacyclic triterpene methyl ethers in New Zealand [START_REF] Connor | Triterpene methyl ether differentiation in Chionochloa (Gramineae)[END_REF].

(iv) Pentacyclic triterpenes are reputed to suffer structural rearrangements and double bond migration during diagenesis that could affect the original diversity of compounds (e.g. [START_REF] Rullkötter | Early diagenesis of terrigenous triterpenoids and its implications for petroleum geochemistry[END_REF]. This phenomenon, well documented from laboratory experiments for compounds the oleanane series [START_REF] Courtney | Triterpenoids of the friedelane series. Part III. The course of friedelane-oleanane rearrangement[END_REF][START_REF] Coates | On the friedelane-oleanane rearrangement[END_REF], is also observed in sedimentary records [START_REF] Rullkötter | Early diagenesis of terrigenous triterpenoids and its implications for petroleum geochemistry[END_REF]. Precisely, friedel-3-ene, glutin-5-ene, multiflor-7-ene and taraxer-14-ene are progressively rearranged to olean-18-ene, olean-12-ene and finally olean-13(18)-ene isomers. Such rearrangements have also been reported for the hopane and fernane series [START_REF] Nishimoto | The structures of arundoin, cylindrin and fernenols. Triterpenoids of fernane and arborane groups of Imperata cylindrical var. koenigii[END_REF] in rock and sediment samples [START_REF] Volkman | Bacterial and algal hydrocarbons in sediments from a saline Antarctic lake, Ace Lake[END_REF][START_REF] Paul | Fernenes and other triterpenoid hydrocarbons in Dicroidium-bearing Triassic mudstones and coals from South Australia[END_REF]. To our knowledge, the only studies about such reactions on triterpenyl acetates that carried out in the laboratory [START_REF] Chatterjee | Lewis acid catalysed rearrangement of triterpenoids[END_REF]1976), showed the acid-induced transformation of taraxeryl acetate to -amyrin acetate, which could further be transformed to -amyrin acetate. To our knowledge, the stability of pichierenyl or swertenyl acetates in natural environments remains to be checked. Thus, in the absence of available evidence, the possibility of rearrangements of triterpenyl acetates in soils can be excluded, further work being needed to ascertain whether this conclusion could be extended to sediments, especially over a long duration.

Potential palaeoenvironmental implications

Few studies have reported the occurrence of triterpenyl acetates in soil and sediments [START_REF] Logan | Preservation of fossil leaf waxes in association with their source tissues, Clarkia, northern Idaho, USA Clarkia, northern Idaho, USA[END_REF][START_REF] Van Bergen | Organic geochemical studies of soils from the Rothamsted Classical Experiments--l. Total lipid extracts, solvent insoluble residues and humic acids from Broadbalk Wilderness[END_REF][START_REF] Xu | Oxygenated spiro-triterpenoids possibly related to arborenes in sediments of a tropical, freshwater lake[END_REF][START_REF] Oyo-Ita | Occurrence and sources of triterpenoid methyl ethers and acetates in sediments of the Cross-River system, Southeast Nigeria[END_REF][START_REF] Trendel | Molecular characterisation of soil surface horizons with different vegetation in the Vosges Massif (France)[END_REF]. According to [START_REF] Oyo-Ita | Occurrence and sources of triterpenoid methyl ethers and acetates in sediments of the Cross-River system, Southeast Nigeria[END_REF], this lack can be explained by the procedures generally applied, saponification entailing loss of the acetate group. These authors also underline the possibility of natural hydrolysis of the ester bond during riverine transport. They therefore attribute the presence of triterpenyl acetates in sediments to a local source. Despite the few reports, no origin was proposed for the compounds, probably because, except for lupeyl acetates, those detected (taraxeryl, germanicyl, -amyrin, -amyrin acetates) were not of direct taxonomical interest. To our knowledge, our work constitutes the first report on bauerane, taraxastane, swertane and pichierane acetates in soil, which can be unequivocally linked to Asteraceae.

Asteraceae are defined as meadow and pasture species (semi-open to open habitats) that colonize disrupted environments and are able to invade fallow land developed on abandoned arable land [START_REF] Cronquist | Vascular Flora of the Southeastern United States[END_REF]Bouby andBillaud, 2001, 2005;[START_REF] Antonetti | Atlas de la Flore d'Auvergne[END_REF]. Their pollen is frequently used as an anthropogenic indicator because it is found mainly in settlements next to cultivated fields, in mowed or grazing places, or in ruderal communities (i.e. growing in rubbles; [START_REF] Hicks | Modern pollen deposition and its use in interpreting the occupation history of the island Hailuoto, Finland[END_REF][START_REF] Court-Picon | Modern pollen/vegetation/land use relationships in mountain environments: an example from the Champsaur valley (French Alps)[END_REF]. Thus, the detection of triterpenyl acetates in soil and sediments could attest to both open vegetation and major perturbation in the catchment, under natural but more obviously anthropogenic influence.

The compounds could constitute diagnostic tracers of agriculture and other human activities. For example, the presence of various Asteraceae seeds in a Late Bronze Age (905-869 BC) settlement on the shore of Lake le Bourget was related to their ethnobotanical properties, their consumption as human food or livestock fodder, dye and/or medicine [START_REF] Bouby | Economie agraire à la fin de l'âge du Bronze sur les bords du Lac du Bourget (Savoie, France)[END_REF]2005). Triterpenyl acetates thus extend the panel of biomarkers for past flora that can attest, in favourable cases, to human activities such as miliacin for millet [START_REF] Jacob | Millet cultivation history in the French Alps as evidenced by a sedimentary molecule[END_REF], other pentacyclic triterpene methyl ethers for Gramineae [START_REF] Ohmoto | Triterpenoids of the Gramineae[END_REF][START_REF] Jacob | Pentacyclic triterpene methyl ethers in recent lacustrine sediments (Lagoa do Caçó, Brazil)[END_REF][START_REF] Zocatelli | Molecular evidence for recent turf cultivation in Northeast Brazil (Lagoa do Boqueirão, RN State)[END_REF], iso-and anteiso-monomethyl alkanes for culinary and aromatic herbs from the Lamiaceae family [START_REF] Huang | Significance of long chain iso and anteiso monomethyl alkanes in the Lamiaceae (mint family)[END_REF] and more generally those reported by [START_REF] Evershed | Organic residue analysis in archaeology: The archaeological biomarker revolution[END_REF] with respect to archaeology. As with these other biomarkers, triterpenyl acetates can constitute a reliable tool for tracing the presence of plants of economic interest.

They could reinforce other widely used techniques that allow detection of the presence of Asteraceae in any sedimentary or archaeological context: for example, for palynological studies that suffer from the under representation of Asteraceae in pollen records because of their dispersal mode [START_REF] Andrieu | Les distorsions de l'enregistrement pollinique de l'histoire de la végétation du dernier cycle climatique: exemples de séquences lacustres du Sud de la France[END_REF].

Conclusions

Neutral lipids in soils developed under pasture and meadow exhibit a wide array of pentacyclic triterpene structures bearing an acetate group at C-3. Among these, C-3 acetates with bauerane, taraxastane, swertane and pichierane skeletons are reported in natural archives for the first time. An exhaustive review of the phytochemical literature allowed us to unequivocally attribute some of the triterpenyl acetates to a single vegetation source within the Asteraceae family. This specificity confers to these compounds a high potential as tracers of Asteraceaeand thus of open environmentsfor palaeoenvironmental studies. Table 1. The spectra are provided in Fig. 2. Fig. 2. Mass spectra and structure of compounds in soil sample. Numbers refer to Table 1 and Fig. 1. Mass spectra of glutinyl, -amyrin and lupeyl acetates were recorded for the standard compound because of GC co-elution with the soil sample.

Figure captions

(

  ii) The compounds are produced by plants other than Asteraceae in the catchment. If the two preceding examples can help clarify the presence of taraxeryl, -amyrin and multiflorenyl acetates, they cannot account for the presence of glutinyl acetate.

Fig. 1 .

 1 Fig. 1. Example of distribution of triterpenyl acetates in ketone/acetate fraction from a

Acknowledgments

The study was supported by the Erode project funded by the INSU/CNRS and Otarie project funded by the Région Centre and the European Council. M.L. also acknowledges the Région Centre for a PhD grant. We thank S. Abrams and K. Nelson (National Research Council of Canada's Plant Biotechnology Institute, Canada) for providing reference compounds from the Professor Tulloch collection, M. Benkhaled (Batna University, Algeria) for multiflorenyl acetate and Professor A. Salatino (São Paulo University, Brazil) for bauerenyl acetate. We also greatly appreciate the help provided by Professor W. Berensohn (Botanical Garden and Botanical Museum, Berlin, Germany), project director of the Bohlmann Files. We are grateful to H.M. Tablbot and two anonymous reviewer for corrections and constructive comments.

Carduus nutans X

Jordon-Thaden and Louda, 2003

Cirsium arvense X X X X X [START_REF] Dutta | Taraxasterol and its derivatives from Cirsium arvense[END_REF][START_REF] Tulloch | Epicuticular wax of Cirsium arvense[END_REF]Jakupovic et al., 2011 Hieracium pilosella L. X Jakupovic et al., 2011 Onopordum acanthium X [START_REF] Khalilova | PMR and 13 C NMR spectra of biologically active compounds. XII. Taraxasterol and its acetate from the aerial part of Onopordum acanthium[END_REF][START_REF] Khalilova | Isolation and crystal structure of taraxasteryl acetate from Onopordum acanthium[END_REF][START_REF] Shoeb | Two salonitenolide derivatives from the aerial parts of Centaurea gigantea inhibit the growth of colorectal cancer cells in vitro[END_REF] Picris echioides X Jakupovic et al., 2011 Picris hieracioides X X X X X X X X X X X Shiojima et al., 1989a; b; 1995; Jakupovic et al., 2011 Solidago virga-aurea X [START_REF] Choi | Phytochemical constituents of the aerial parts from Solidago virga-aurea var. gigantean[END_REF] Sonchus oleraceus L. X X X Jakupovic et al., 2011 Taraxacum officinale X [START_REF] Hänsel | Sesquiterpenlacton--D-glucopyranoside sowie ein neues eudesmanolid ais Taraxacum officinale[END_REF] Total (a) X X X X X X X X X X X (b) Asteraceae genera Artemisia (2) X X X X X X [START_REF] Lao | Studies on the constituents of Artemisia rubripes Nakai[END_REF][START_REF] Cranwell | Organic geochemistry of lacustrine sediments: triterpenoids of higher plant origin reflecting post-glacial vegetational succession[END_REF][START_REF] Yang | Chemical constituents of Artemisia dalailamae Kraschen[END_REF]Jakupovic et al., 2011 Cirsium (7) X X X X X X [START_REF] Dutta | Taraxasterol and its derivatives from Cirsium arvense[END_REF][START_REF] Ulubelen | Triterpenic and steroidal compounds of Cnicus Benedictus[END_REF][START_REF] Tulloch | Epicuticular wax of Cirsium arvense[END_REF]Jakupovic et al., 2011 Inula (1) X X X [START_REF] Ahmad | 5-Hydroxy-3,6,7,2',5'-pentamethoxyflavone from Inula grantioides[END_REF]Jakupovic et al., 2011 Total

van [START_REF] Van Bergen | Organic geochemical studies of soils from the Rothamsted Classical Experiments--l. Total lipid extracts, solvent insoluble residues and humic acids from Broadbalk Wilderness[END_REF][START_REF] Ding | Triterpene compounds isolated from Acer mandshuricum and their anti-inflammatory activity[END_REF] Euphorbia X X X X X X X X [START_REF] Dutta | Taraxasterol and its derivatives from Cirsium arvense[END_REF][START_REF] Ahmad | Chemical constituents of Euphorbia granulaia[END_REF][START_REF] Liu | Studies on chemical constituents from Euphorbia fischeriana Steud[END_REF][START_REF] Shi | A new bisabolane sesquiterpenoid from Euphorbia chrysocoma[END_REF][START_REF] Haba | Diterpenoids and triterpenoids from Euphorbia guyoniana[END_REF] Total (a)+(b)+(c) X X X X X X X X X X X X X X X a (a) Asteraceae species in Lake Aydat catchment; (b) Asteraceae genera in which triterpenyl acetates have been described; b numbers in parentheses refer to number of species of a given genera described in the catchment according to [START_REF] Antonetti | Atlas de la Flore d'Auvergne[END_REF].


Patricia Gibert
email: patricia.gibert@univ-lyon1.fr

Vincent Debat

Cameron K Ghalambor

Phenotypic plasticity, global change, and the speed of adaptive evolution

Highlights :

3 to 5 bullet points (maximum 85 characters, including spaces, per bullet point)

-Phenotypic plasticity can influence the rate of evolution of insects -The rate of evolution will depend on how plasticity alters the strength of selection -The response to selection will also depend on plasticity alters trait correlations -More studies are needed to address this question in insects

Abstract (100-200 mots)

Faced with a rapidly changing environment, phenotypic plasticity allows for the production of a diversity of intra-generational responses, but the subsequent consequence inter-generational evolutionary processes is still difficult to predict. In this article, we review theory and empirical studies addressing this question in insects by considering three scenarios. Adaptive plasticity that should lead to slow or no evolution. Non-adaptive phenotypic plasticity to new environmental conditions that should lead either to extinction or, on the contrary, to rapid evolutionary change.

The third scenario deals with how plasticity alters the variance selection acts upon. These scenarios are then discussed by highlighting the effects of genetic correlations between phenotypes. We conclude that more studies are needed in insects to better understand the relationship between phenotypic plasticity and evolutionary processes.

Introduction

The first response of organisms to environmental change is often through plasticity in behavior, physiology, or other environmentally sensitive traits [START_REF] Price | The role of phenotypic plasticity in driving genetic evolution[END_REF][START_REF] Paenke | Influence of Plasticity and Learning on Evolution under Directional Selection[END_REF][START_REF] Ghalambor | Adaptive versus non-adaptive phenotypic plasticity and the potential for contemporary adaptation in new environments[END_REF]. Such plasticity is by definition a non-evolutionary response to the environment, because it occurs within the generation of an individual. However, a long-standing question in evolutionary biology is whether the plasticity observed within generations influences subsequent evolutionary change across generations [START_REF] Price | The role of phenotypic plasticity in driving genetic evolution[END_REF][START_REF] Paenke | Influence of Plasticity and Learning on Evolution under Directional Selection[END_REF][START_REF] Ghalambor | Adaptive versus non-adaptive phenotypic plasticity and the potential for contemporary adaptation in new environments[END_REF]. Understanding the relationship between plasticity and evolution has taken on increased importance in the context of rapid global change as historic patterns of temperature and rainfall are simultaneously changing in a directional manner and showing increased variability [START_REF] Hoffmann | Climate change and evolutionary adaptation[END_REF][START_REF] Sgrò | What Can Plasticity Contribute to Insect Responses to Climate Change?[END_REF].

Insects, like other ectotherms exhibit plasticity in a suite of traits related to temperature (reviewed in [START_REF] Sgrò | What Can Plasticity Contribute to Insect Responses to Climate Change?[END_REF]), and because of their relatively fast generation times also have the potential to rapidly evolve [START_REF] Loxdale | Rapid genetic changes in natural insect populations[END_REF]. Insect populations are therefore good models for considering the relationship between plasticity and evolution. For example, the life cycles of insects living at higher latitudes have evolved plastic responses to specific environmental cues that help them survive cold winters, but climate change is increasingly disrupting the reliability of these cues [START_REF] Sgrò | What Can Plasticity Contribute to Insect Responses to Climate Change?[END_REF]. How might this disruption alter patterns of plasticity and subsequent evolutionary change? Our goal here is to provide possible answers to this question by reviewing evolutionary theory and presenting different scenarios for how plasticity will alter the rate of adaptive evolution. Where possible, we use empirical studies of insects as examples to illustrate these concepts.

The evolutionary response of a trait to an episode of selection is dependent on the strength of directional selection and the amount of genetic variance [START_REF] Lande | The Measurement of Selection on Correlated Characters[END_REF]. Thus, understanding how plasticity impacts the heritability or additive genetic variance/covariance for a trait, and the strength of selection should allow for predicting the rate of evolutionary change in a population. Indeed, a traditional perspective is that plasticity slows the rate of evolutionary change because most of the phenotypic variance is environmentally induced and non-heritable [START_REF] Ghalambor | Adaptive versus non-adaptive phenotypic plasticity and the potential for contemporary adaptation in new environments[END_REF]. Under such conditions even strong selection will fail to produce an evolutionary response. However, it is now generally accepted that plasticity is itself a trait that exhibits genetic variation and has the capacity to evolve [START_REF] Scheiner | Genetics and Evolution of Phenotypic Plasticity[END_REF]. If plasticity itself is heritable, then a critical question becomes how a particular environment alters the distribution of phenotypes within a population relative to the optimal phenotype (e.g. [START_REF] Price | The role of phenotypic plasticity in driving genetic evolution[END_REF][START_REF] Paenke | Influence of Plasticity and Learning on Evolution under Directional Selection[END_REF][START_REF] Ghalambor | Adaptive versus non-adaptive phenotypic plasticity and the potential for contemporary adaptation in new environments[END_REF].). We focus on three scenarios: 1) adaptive plasticity, where the environment predictably shifts the distribution of phenotypes towards the optimum, 2) nonadaptive plasticity, where the environment predictably shifts the distribution of phenotypes away from the optimum, and 3) effect on variance, where the environment alters the range of variance available for selection to act on (reviewed in [START_REF] Ghalambor | Adaptive versus non-adaptive phenotypic plasticity and the potential for contemporary adaptation in new environments[END_REF]). Below we expand on these ideas in more detail and the implications for insects dealing with global change.

I Adaptive Plasticity

Adaptive plasticity is used here in the sense of beneficial plasticity, i.e. that allows individuals to have a higher fitness in the new environment than it would be if not plastic [START_REF] Ghalambor | Adaptive versus non-adaptive phenotypic plasticity and the potential for contemporary adaptation in new environments[END_REF][START_REF] Gotthard | Adaptive Plasticity and Plasticity as an Adaptation: A Selective Review of Plasticity in Animal Morphology and Life History[END_REF][START_REF] Schmid | The role of phenotypic plasticity on population differentiation[END_REF]. There are many examples of adaptive phenotypic plasticity in insects. In butterfly species, seasonal plasticity (i.e. polyphenism - [START_REF] Shapiro | Seasonal polyphenism[END_REF]) with regard to adult colour pattern [START_REF] Windig | Reaction norms and the genetic basis of phenotypic plasticity in the wing pattern of the butterfly Bicyclus anynana[END_REF][START_REF] Koch | Ecdysteroids control eyespot size and wing color pattern in the polyphenic butterfly Bicyclus anynana (Lepidoptera: Satyridae)[END_REF][START_REF] Roskam | Lepidoptera : Satyridae ) butterflies : different climates need different cues[END_REF][START_REF] Mateus | Adaptive developmental plasticity: Compartmentalized responses to environmental cues and to corresponding internal signals provide phenotypic flexibility[END_REF] has received considerable attention and was shown to be adaptive [START_REF] Kingsolver | Fitness consequences of seaonal polyphenism in westen white butterflies[END_REF][START_REF] Brakefield | Seasonal polyphenism in butterflies and natural selection[END_REF][START_REF] Kemp | Phenotypic plasticity in field populations of the tropical butterfly Hypolimnas bolina ( L .) ( Nymp halidae )[END_REF][START_REF] Hazel | The environmental and genetic control of seasonal polyphenism in larval color and its adaptive significance in a swallowtail butterfly[END_REF][START_REF] Stoehr | Seasonal phenotypic plasticity of wing melanisation in the cabbage white butterfl y , Pieris rapae L . ( Lepidoptera : Pieridae )[END_REF]. Another well documented example is the case of diapause induction, the cessation of reproduction and/or suppressed metabolism as a strategy to persist through harsh conditions (reviewed in [START_REF] Sgrò | What Can Plasticity Contribute to Insect Responses to Climate Change?[END_REF]).

In the event of a rapid change in the environment, adaptive phenotypic plasticity is hypothesized to allow organisms to 1) adaptively modify traits over a very short (intragenerational) timescale, 2) assist in the initial survival of populations, and 3) reduce the probability of extinction [START_REF] Pigliucci | Phenotypic plasticity. Beyond nature and nurture[END_REF]. While this hypothesis has been the subject of much modelling work [START_REF] Lande | Adaptation to an extraordinary environment by evolution of phenotypic plasticity and genetic assimilation[END_REF][START_REF] Chevin | Adaptation, Plasticity, and Extinction in a Changing Environment: Towards a Predictive Theory[END_REF][START_REF] Ashander | Predicting evolutionary rescue via evolving plasticity in stochastic environments[END_REF][25][START_REF] Nunney | Adapting to a changing environment: modeling the interaction of directional selection and plasticity[END_REF], it has received very little empirical evidence at this time. However, comparisons of phenotypic plasticity in invasive and native populations have been used to address this question. Such studies are abundant in plants with mixed results [START_REF] Richards | Jack of all trades, master of some? On the role of phenotypic plasticity in plant invasions[END_REF]. In insects, the degree to which adaptive plasticity benefits to the first step of the invasion starts to be studied in Drosophilids [START_REF] Gibert | Drosophila as models to understand the adaptive process during invasion[END_REF] and in particular on the morphology of the wing, a very temperature sensitive trait that can evolve quickly and is probably adaptively [START_REF] Huey | Rapid evolution of a geographic cline in size in an introduced fly[END_REF][START_REF] Nunney | The Effect of Temperature on Body Size and Fecundity in Female Drosophila melanogaster: Evidence for Adaptive Plasticity[END_REF]. For example, Loh et al [START_REF] Loh | Adaptation to different climates results in divergent phenotypic plasticity of wing size and shape in an invasive drosophilid[END_REF] found significantly different plastic responses to temperature in the wing size of Brazilian vs African populations of Zaprionus indianus. Fraimout et al. [START_REF] Fraimout | Phenotypic plasticity of Drosophila suzukii wing to developmental temperature: implications for flight[END_REF] found that wing plasticity was associated to an effect on flight speed but did not find any significant differences in wing morphology plasticity between three populations of Drosophila suzukii.

How should adaptive plasticity influence the rate of adaptive evolution? If the phenotype is close to the optimum, then directional selection will be weak and the population should evolve slowly or not at all unless there is a substantial fitness cost to plasticity [START_REF] Price | The role of phenotypic plasticity in driving genetic evolution[END_REF][START_REF] Ghalambor | Adaptive versus non-adaptive phenotypic plasticity and the potential for contemporary adaptation in new environments[END_REF][START_REF] Crispo | Modifying effects of phenotypic plasticity on interactions among natural selection, adaptation and gene flow[END_REF]. Quantitative genetic approaches [START_REF] Lande | The Measurement of Selection on Correlated Characters[END_REF] can be used to determine the optimum phenotype and the strength of selection. Another strategy is to compare the reaction norms of populations occupying different environments. If adaptive phenotypic plasticity weakens selection, then there should be little genetic differentiation between populations. For example, Ayrinhac et al. [START_REF] Ayrinhac | Cold adaptation in geographical populations of Drosophila melanogaster: phenotypic plasticity is more important than genetic variability[END_REF] compared populations of Drosophila melanogaster for chill coma recovery time, a very plastic trait depending on developmental temperature, and found the adaptive variation arose from phenotypic plasticity, not genetic differentiation. Similar conclusions have been obtained on other traits in Drosophila [START_REF] Gibert | Chill-coma temperature in Drosophila: effects of developmental temperature, latitude, and phylogeny[END_REF][START_REF] Liefting | Plasticity versus environmental canalization: population differences in thermal responses along a latitudinal gradient in Drosophila serrata[END_REF][START_REF] Klepsatel | Variation in thermal performance and reaction norms among populations of drosophila melanogaster[END_REF][START_REF] Hoffmann | Relative importance of plastic vs genetic factors in adaptive differentiation: geographical variation for stress resistance in Drosophila melanogaster from eastern Australia[END_REF] and in some other species [START_REF] Wool | Phenotypic Plasticity in Australian Cotton Aphid ( Homoptera : Aphididae ): Host Plant Effects on Morphological Variation[END_REF]40] but in many studies, this question is generally not specifically addressed, especially in the case of non-linear reaction norm.

II. Non-Adaptive Phenotypic Plasticity

While natural selection has led to the evolution of adaptive plasticity, not all plastic responses to the environment are adaptive. We define non-adaptive plasticity as an environmentally-induced change that shifts the phenotype further away from the phenotypic optimum [START_REF] Ghalambor | Adaptive versus non-adaptive phenotypic plasticity and the potential for contemporary adaptation in new environments[END_REF]41]. Under such a definition, non-adaptive plasticity is not beneficial in terms of individual fitness. At the population level, the presence of non-adaptive plasticity is most likely to be found under novel environmental conditions where past selection has not had an opportunity to act on the genetic variation for plasticity [START_REF] Ghalambor | Adaptive versus non-adaptive phenotypic plasticity and the potential for contemporary adaptation in new environments[END_REF]. Non-adaptive plasticity has two potential evolutionary outcomes. The first potential outcome is the extinction of the population. Extinction is always a possible outcome in response to a rapid or strong episode of selection, and any non-adaptive plastic response should exacerbate this possibility, particularly if the trait(s) involved are closely tied to fitness [START_REF] Chevin | Adaptation, Plasticity, and Extinction in a Changing Environment: Towards a Predictive Theory[END_REF]. A second potential outcome is rapid adaptive evolution (e.g. [START_REF] Ghalambor | Non-adaptive plasticity potentiates rapid adaptive evolution of gene expression in nature[END_REF]. Non-adaptive plasticity can facilitate rapid evolution by increasing the strength of selection, and given the presence of sufficient genetic variation, lead to adaptive changes in either the trait or the reaction norm of the trait [START_REF] Ghalambor | Non-adaptive plasticity potentiates rapid adaptive evolution of gene expression in nature[END_REF][START_REF] Chevin | Phenotypic plasticity in evolutionary rescue experiments[END_REF]. In such a scenario, non-adaptive plasticity may be a transient phenomenon that is important during the early stages of adaptation when the difference between the plastic response and the local optimum are greatest and the strength of selection strongest. However, determining what the local optimum for any phenotype is, and the degree to which any given environment is really novel are challenging problems when attempting to assess non-adaptive phenotypic plasticity.

In the context of global change, the critical question is: how often might insect populations exhibit non-adaptive plasticity? In theory, two scenarios are expected to generate non-adaptive plastic responses to the environment: 1) countergradient selection, and 2) extreme environmental conditions (heatwaves, prolonged droughts, cold-snaps, etc..) that lie outside the historic range of environmental conditions. Countergradient selection describes situations where the plastic response to the environment is non-adaptive or in the opposite direction favored by selection, resulting in genetic compensation or cryptic adaptation [START_REF] Conover | Phenotypic similarity and the evolutionary significance of countergradient variation[END_REF][START_REF] Conover | The Covariance between Genetic and Environmental Influences across Ecological Gradients Reassessing the Evolutionary Significance of Countergradient and Cogradient Variation[END_REF][START_REF] Grether | Countergratient variation in the sexual coloration of guppies (Poecilia reticulata): Drosopterin synthesis balances carotenoid availability[END_REF]. Conover et al. [START_REF] Conover | The Covariance between Genetic and Environmental Influences across Ecological Gradients Reassessing the Evolutionary Significance of Countergradient and Cogradient Variation[END_REF] review such cases and find numerous insect examples across temperature gradients associated with latitude or elevation. For example, high latitude insect populations are under selection for faster growth rates in response to colder temperature, but the plastic response to cold temperatures is slower growth leading to adaptive evolution for faster growth rates in high relative to low latitude populations (reviewed in [START_REF] Conover | The Covariance between Genetic and Environmental Influences across Ecological Gradients Reassessing the Evolutionary Significance of Countergradient and Cogradient Variation[END_REF]). Extreme environments outside the range of historic conditions may also generate non-adaptive plasticity because selection has not had the opportunity to shape the plastic response (e.g. [START_REF] Ghalambor | Adaptive versus non-adaptive phenotypic plasticity and the potential for contemporary adaptation in new environments[END_REF]). Insects may be particularly sensitive to such extreme climate events, particularly at high latitudes where populations have evolved physiological and lifehistory traits to survive cold winters (see [START_REF] Sgrò | What Can Plasticity Contribute to Insect Responses to Climate Change?[END_REF]). For example, warmer autumn temperatures may not sufficiently prepare insects physiologically to survive cold winter temperatures [START_REF] Coleman | Cross-generation plasticity in cold hardiness is associated with diapause, but not the non-diapause developmental pathway, in the blow fly Calliphora vicina[END_REF][START_REF] Stuhldreher | Cold-adapted species in a warming world -an explorative study on the impact of high winter temperatures on a continental butter fl[END_REF], while warmer winter temperatures may cause non-adaptive early emergence from diapause leading to higher mortality [START_REF] Stuhldreher | Cold-adapted species in a warming world -an explorative study on the impact of high winter temperatures on a continental butter fl[END_REF][START_REF] Bale | Insect overwintering in a changing climate[END_REF].

III. Stressful environmental conditions and phenotypic variation

A classic tenet of ecological genetics -largely based upon laboratory studies on Drosophila -is that stressful environmental conditions tend to increase phenotypic variation [START_REF] Hoffmann | Extreme Environmental Change and Evolution[END_REF][START_REF] Hoffmann | Evolutionary genetics and environmental stress[END_REF]. As adaptation depends on the existence of heritable variation (R = h 2 S, where R is the response to selection, S is the selection differential and h 2 is heritability), this effect might have deep evolutionary consequences, and in particular it may influence the adaptability to ongoing global changes. In the previous examples of adaptive and non-adaptive plasticity we focused largely on changes in S. However, stressful conditions can also potentially induce increased heritability (h 2 =V A /V P , where V A is additive genetic variance and V P the total phenotypic variance), if the increased phenotypic variance is genetically based. A higher heritability and response to selection could boost the adaptive potential to the new stressful conditions. Alternatively, if the increased variance is not genetically based, an increase in V P would in turn decrease heritability, reducing the evolutionary potential. The generality of the stress-induced increase in variation has nevertheless been seriously questioned in the past two decades [START_REF] Charmantier | Environmental quality and evolutionary potential: Lessons from wild populations[END_REF][START_REF] Hoffmann | Heritable variation and evolution under favourable and unfavourable conditions[END_REF].

Can we identify a general effect of stress upon genetic variation and what can insects studies tell us about it?

The interest for stress-induced variation may c ck W ' xp with Drosophila [START_REF] Waddington | Canalization of development and the inheritance of acuired characters[END_REF]. He showed that stressors such as heat shocks or ether vapor could elicit a burst of phenotypic variation upon which artificial selection could be successfully applied. The evolutionary response demonstrated that the elicited variation was indeed genetic -but conditional to the environmental stress. Such storage and release of cryptic genetic variation and their molecular bases have since received much attention (e.g. [START_REF] Gibson | Drosophila Regulatory Gene Ultrabithorax on Homeotic Stability[END_REF][START_REF] Rutherford | Hsp90 as a capacitor for morphological evolution[END_REF][START_REF] Gibson | Uncovering cryptic genetic variation[END_REF][START_REF] Paaby | Cryptic genetic variation: Evolution's hidden substrate[END_REF]). In particular, the work on Hsp90 in Drosophila [START_REF] Rutherford | Hsp90 as a capacitor for morphological evolution[END_REF] has shown that a wide range of cryptic genetic variants could be stored and conditionally released. The induced variation is however generally detrimental (Figure 1B; [START_REF] Rutherford | Hsp90 as a capacitor for morphological evolution[END_REF][START_REF] Wagner | Is Hsp90 a regulator of evolvability?[END_REF][START_REF] Masel | Cryptic genetic variation is enriched for potential adaptations[END_REF]) and whether it could contribute to adaptive evolution has been controversial (e.g. [START_REF] Mcguigan | Evolutionary consequences of cryptic genetic variation[END_REF]).

The hypothesis that stress induces an increase in genetic variation is not universally supported. Hoffmann and Merilä [START_REF] Hoffmann | Heritable variation and evolution under favourable and unfavourable conditions[END_REF] were the first to challenge the generality of this hypothesis. Later, Charmantier and Garant [START_REF] Charmantier | Environmental quality and evolutionary potential: Lessons from wild populations[END_REF] reported in a meta-analysis that heritability rather tends to increase under favorable conditions. This discrepancy with the early Drosophila laboratory studies was discussed in terms of environmental novelty. In natural populations, stressful conditions would be the norm rather than the exception, while the stressors exerted in lab conditions would often represent completely new conditions. Many studies since then have explored the effects of extreme and novel environmental conditions on genetic variation for various traits, producing contrasting results. Among insects, a few Drosophila studies have reported an increase in genetic variation with stressful temperature [START_REF] Swindell | Reduced inbreeding depression due to historical inbreeding in Drosophila melanogaster: Evidence for purging[END_REF][START_REF] Sisodia | Variations in morphological and life-history traits under extreme temperatures in Drosophila ananassae[END_REF][START_REF] Van Heerwaarden | Increases in the evolutionary potential of upper thermal limits under warmer temperatures in two rainforest Drosophila species[END_REF] or larval crowding [START_REF] Horváth | Effects of larval crowding on quantitative variation for development time and viability in Drosophila melanogaster[END_REF]. High temperature was found to reveal cryptic genetic variation in a female sperm storage organ in a sepsid fly [START_REF] Berger | Experimental evolution for generalists and specialists reveals multivariate genetic constraints on thermal reaction norms[END_REF]. Drought stress increased additive genetic variation for wing size and melanization in a butterfly species [START_REF] Talloen | Environmental stress and quantitative genetic variation in butterfly wing characteristics[END_REF]. Edvardsson et al [START_REF] Edvardsson | Quantitative genetic variation in the control of ovarian apoptosis under different environments[END_REF] detected a strong increase in additive genetic variation for ovarian apoptosis in starved cockroach females. In contrast, other studies failed to detect such effect: for example, in Drosophila, genetic variation for heat resistance was not affected by drought stress [START_REF] Bubliy | Humidity affects genetic architecture of heat resistance in Drosophila melanogaster[END_REF] and neither was genetic variation for viability by temperature [START_REF] Ketola | Constant, cycling, hot and cold thermal environments: Strong effects on mean viability but not on genetic estimates[END_REF]. A recent meta-analysis based on 39 studies (including 11 on insects) suggests no consistent effect of stressful conditions for morphology, while life history traits tend to present higher genetic variation under stress [START_REF]Environmental stress correlates with increases in both genetic and residual variances: A meta-analysis of animal studies[END_REF]. Altogether, these results suggest that although genetic variation is clearly dependent on the environment, the effect of stress is likely trait, taxon and environment specific.

Discussion

The exact relationship between plasticity and evolution can be difficult to predict, but our perspective here is that by focusing on how plasticity alters the strength of selection and the amount of genetic variation expressed, predictions can be made about the rate of evolution. We summarize this perspective in a pair of conceptual figures (Figures 1 &2). We can conceptualize the relationship between plasticity and adaptive evolution using a modified view of Fisher [START_REF] Fisher | The genetical theory of natural selection[END_REF] O ' [START_REF] Orr | The Population Genetics of Adaptation: The Distribution of Factors Fixed during Adaptive Evolution[END_REF] geometric model of adaptation (Figure 1). Rather than considering the distributional effects of mutations on the adaptive walk towards a local optimum, we can instead consider the effects of plasticity on a fitness landscape (following [START_REF] Ghalambor | Adaptive versus non-adaptive phenotypic plasticity and the potential for contemporary adaptation in new environments[END_REF]). Collectively, the perspective presented here argues that predicting if insects will evolve in response to climate or any other environmental change requires qualitatively or quantitatively estimating how the plastic responses alter the strength of selection. We recognize this is challenging, but not impossible. For example, numerous studies to date have asked whether patterns of plasticity in thermal tolerance are adaptive and sufficient to buffer populations under future climate scenarios [START_REF] Sgrò | What Can Plasticity Contribute to Insect Responses to Climate Change?[END_REF].

The scenarios depicted in Fig. 1 assume plasticity as a univariate response, however, adaptation to changing environments is more likely to be a multivariate process involving numerous genetically correlated traits. The G-matrix or genetic variance-covariance matrix is a statistical tool for quantifying trait correlations [START_REF] Lande | The Measurement of Selection on Correlated Characters[END_REF][START_REF] Lande | Quantitative Genetic Analysis of Multivariate Evolution , Applied to Brain : Body Size Allometry[END_REF] and can be visualized as an ellipse [START_REF] Steppan | Comparative quantitative genetics: evolution of the G matrix[END_REF][START_REF] Schluter | Adaptive Radiation Along Genetic Lines of Least Resistance[END_REF], whose axes represent the main directions of available genetic variation. Such trait correlations can either constrain the evolutionary response to selection if the selection gradient is orthogonal to the major axis of variation or facilitate the evolutionary response if selection is along the major axis [START_REF] Steppan | Comparative quantitative genetics: evolution of the G matrix[END_REF][START_REF] Schluter | Adaptive Radiation Along Genetic Lines of Least Resistance[END_REF]. However, there is also considerable evidence that trait correlations are plastic [START_REF] Andersen | How to assess Drosophila cold tolerance: chill coma temperature and lower lethal temperature are the best predictors of cold distribution limits[END_REF][START_REF] Wood | Environmental effects on the structure of the G-matrix[END_REF][START_REF] Sgrò | Genetic correlations, tradeoffs and environmental variation[END_REF], and thus could influence the evolutionary response to selection. We depict some of these outcomes in Figure 2, using a stable fitness landscape as in Figure 1.

In summary, phenotypic plasticity in a diversity of traits will be the first response of organisms to environmental change, and these plastic responses will in turn have implications for how populations evolve by altering the strength of selection and amount of expressed genetic variation. Yet, few studies have explored the linkages between plasticity and evolution in natural populations of insects. We argue that more studies should explicitly test such linkages to better improve long-term projections of how insect populations will perform under future climate scenarios.

Figure Legends

Figure 1 -When adaptive plasticity results in a perfect match with the optimum (Fig. 1a1), there is no opportunity for directional selection and thus no evolutionary change is predicted. When adaptive plasticity reduces the mismatch between the phenotype produced and the optimum (Fig. 1a2), there is an opportunity for directional selection can act, but we would predict a weaker evolutionary response and the rate of adaptive evolution to be slower. In contrast, because nonadaptive plasticity increases the mismatch between the expressed phenotype and the optimum (Figure 1a3), the strength of directional selection is increased and should result in either rapid adaptive evolution or extinction. In cases where the environment induces a release of genetic variation (Figure 1b) we expect a range of non-adaptive and adaptive responses and selection to favor those genotypes that produce the most adaptive responses.

Figure 2 -Considering a population where the major axis of genetic variation is orthogonal to the direction of the local optimum (Figure 2a), we would predict the evolutionary response to be slow and follow a trajectory that is biased by the major axis. In contrast, plasticity in trait correlations can lead to diversity of outcomes that might influence evolutionary responses. For example, plasticity could re-orient the major axis towards the phenotypic optimum, facilitating adaptive evolution (Figure 2b-1). Alternatively, plasticity could shift both the orientation and the location of the matrix, such that trait combinations are closer to the local optimum (Figure 2b-2,3), but differ in their orientation (Figure 2b-2,3). Lastly, plasticity may simply increase the variance (Figure 2b-4).

Acknowledgements

This work was supported by the CNRS GD 37 "Ph p c p c ". C.K.G. was supported by the University Lyon 1 and NSF (IOS-1457383).