Cryptic no more: soil macrofossils uncover Pleistocene forest microrefugia within a periglacial...

Cryptic no more: soil macrofossils uncover Pleistocene forestmicrorefugia within a periglacial desert

Guillaume de Lafontaine1,2,3, Carlos Alberto Amasifuen Guerra1,2, Alexis Ducousso1,2 and R�emy J. Petit1,2

1INRA, UMR 1202 BIOGECO, F-33610, Cestas, France; 2Univ. Bordeaux, UMR 1202 BIOGECO, F-33400 Talence, France; 3Canada Research Chair in Forest and Environmental

Genomics, Centre for Forest Research, Institute for Systems and Integrative Biology, Universit�e Laval, 1030 avenue de la M�edecine, Qu�ebec, QC, G1V 0A6, Canada

Author for correspondence:Guillaume de Lafontaine

Tel: +1 418 656 2131 ext. 12328Email: [email protected]

Received: 28 January 2014

Accepted: 2 April 2014

New Phytologist (2014) 204: 715–729doi: 10.1111/nph.12833

Key words: cryptic refugia, Fagus sylvatica(European beech), forest tree refugia, Hein-rich stadial-1, Late Pleniglacial interval, mac-rofossil charcoal, microrefugia, radiocarbondating.

Summary

� Despite their critical importance for understanding the local effects of global climate change

on biodiversity, glacial microrefugia are not well studied because they are difficult to detect by

using classical palaeoecological or population genetics approaches. We used soil macrofossil

charcoal analysis to uncover the presence of cryptic glacial refugia for European beech (Fagus

sylvatica) and other tree species in the Landes de Gascogne (southwestern France).� Using botanical identification and direct radiocarbon dating (140 14C-dates) of macrofossil

charcoal extracted from mineral soils, we reconstructed the glacial and postglacial history of

all extant beech stands in the region (n = 11).� Soil charcoal macrofossils were found in all sites, allowing the identification of up to at least

14 distinct fire events per site. There was direct evidence of the presence of beech during the

last glacial period at three sites. Beech was detected during Heinrich stadial-1, one of the cold-

est and driest intervals of the last glacial period in Western Europe.� Together with previous results on the genetic structure of the species in the region, these

findings suggest that beech persisted in situ in several microrefugia through full glacial and

interglacial periods up to the present day.

Introduction

Temperate and boreal taxa persisted in refugia during glacialpeaks such as the Late Pleniglacial (LPG) interval, between c. 24and 14.6 kyr BP (Genty et al., 2005a; Tzedakis et al., 2013). Somebut not all of these refugia formed the starting point for expan-sion into newly climatically suitable areas (Huntley & Webb,1989; Bennett et al., 1991; Hewitt, 1996). The traditionalapproaches used to identify refugia rely on pollen stratigraphies(e.g. Davis, 1983; Huntley & Birks, 1983; Ritchie, 1987; Webb,1987; Tzedakis et al., 2002) or on broadscale phylogeographicalsurveys (e.g. Soltis et al., 1997, 2006; Taberlet et al., 1998;Hewitt, 1999, 2000; Petit et al., 2003). In Europe, a general viewarising from these approaches was that the majority of temperateand boreal tree taxa persisted during the LPG interval in threemajor refugial areas within the Mediterranean peninsulas (Ibe-rian, Italian and Balkan), while unglaciated areas north and eastof the southern mountain ranges (Pyrenees, Alps and SouthernCarpathians) were treeless (Huntley & Birks, 1983; Bennettet al., 1991). Recent evidence from megafossils (Kullman, 2002),macrofossils (Willis et al., 2000; Willis & van Andel, 2004; Birks& Willis, 2008; Binney et al., 2009; Kaltenrieder et al., 2009;Haesaerts et al., 2010), phylogeography (e.g. Palm�e et al., 2003;Lascoux et al., 2004; McLachlan et al., 2005; Anderson et al.,2006), and integrated research combining palaeoecology and

genetics (Magri et al., 2006; Parducci et al., 2012; but see Birkset al., 2012) have challenged this theory by suggesting the exis-tence of mid- to high-latitude local refugia outside their main his-torical range, in areas previously considered climaticallyunsuitable for trees during the LPG interval (Stewart & Lister,2001; Bennett & Provan, 2008). Such glacial ‘microrefugia’ wereinitially overlooked when using traditional approaches due tolimited spatial resolution, in contrast to ‘macrorefugia’ – regionswhose general climate was more favourable (Rull, 2009). Cruzan& Templeton (2000) referred to putative microrefugia inferredindirectly from genetic analyses of extant populations as ‘crypticrefugia’, because their exact locations are unknown and prospectsto determine these locations seem very low (see also Stewart &Lister, 2001; Bennett & Provan, 2008; Bhagwat & Willis, 2008;Birks & Willis, 2008; Provan & Bennett, 2008; Rull, 2010;Stewart et al., 2010).

Yet, uncovering the existence of disjunct refugial populationsshould be a priority because they are critical in many respects.They testify that the species range was more extensive during gla-cial maxima than previously thought. Moreover, the demonstra-tion that a species had persisted under very harsh climaticconditions during the last glacial episode suggests a high level ofphenotypic plasticity or adaptive capacity (Bhagwat & Willis,2008; Mosblech et al., 2011), even if local microclimate likelyhelped buffer environmental conditions. Also, small disjunct tree

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populations located at comparatively high latitudes could haveplayed a major role during postglacial recolonization (McLachlanet al., 2005; Anderson et al., 2006; Magri et al., 2006; Magri,2008). This, in turn, would force us to reconsider the dispersalcapacity of species exposed to rapid anthropogenic climate warm-ing (Feurdean et al., 2013). Finally, identifying some of the eco-logical and geographical features that allowed small populationsto persist locally in microrefugia should help clarify how muchdecoupling can exist between local environmental conditions andregional climatic trends, a topic attracting much interest(Ashcroft, 2010; Dobrowski, 2011; Hampe & Jump, 2011;Ashcroft et al., 2012; Keppel et al., 2012).

However, to date, microrefugia remain poorly known despitetheir importance for our understanding of the local effects ofglobal climate change (Noss, 2001; Mosblech et al., 2011; Keppel& Wardell-Johnson, 2012). The difficulty of finding direct fieldevidence of glacial microrefugia has even led some authors toquestion their existence (Birks, 2003; Birks et al., 2005, 2012;Tzedakis et al., 2013). Indeed, palaeoecological reconstructionsbased on fossil pollen analysis alone are ill-suited for identifyingmicrorefugia. Because pollen can originate from long distances, itis problematic to use it to distinguish between local presence atvery low density (microrefugium) and long-distance transportfrom extra-local sources (i.e. from low-latitude macrorefugia)(Birks & Birks, 2000; Willis et al., 2000; Birks, 2003; Willis &van Andel, 2004; Birks & Willis, 2008; Tzedakis et al., 2013). Ithas been proposed that pollen production was very low in thepeak of the glacial period, making it extremely difficult to inter-pret the corresponding signal (Birks & Willis, 2008; Tzedakiset al., 2013). A possible solution is to validate reconstructionsbased on pollen data with plant macrofossil records (e.g. the mul-tiproxy study of Kaltenrieder et al., 2009) because macrofossilsare much less dispersed away from their sources (Birks & Birks,2000; Birks, 2003). However, in contrast with pollen, plant ma-crofossils are usually produced in lower abundance, are not aswell preserved and are harder to identify (Birks & Birks, 2000).Moreover, macrofossil finds are dependent upon their deposi-tional context and suitable preservation environments are limitedto small lake sediments, peatlands and caves, to the point thatentire geographic regions can be completely devoid of suitablesampling sites (Jackson et al., 1997; Cruzan & Templeton, 2000;Bhiry & Filion, 2001; Birks, 2003; Binney et al., 2009). Theavailability of appropriate preservation environments can thusconstitute a limitation to the inferential power of some palaeo-ecological approaches (pollen and especially macrofossil analyses)with respect to our ability to detect in situ microrefugia (but seeBinney et al., 2009).

A complementary palaeoecological approach relies on theanalysis of fossilized wood preserved as macroscopic charcoal inmineral soil. Macrofossil charcoal has been recognized as apromising approach to identify microrefugia for at least five rea-sons (Willis et al., 2000; Stewart & Lister, 2001; Willis & vanAndel, 2004). (1) Ease of taxonomic identification: macrofossilcharcoal represents the remains of burnt trees or shrubs that canbe identified with remarkable accuracy using microscopic woodanatomy (Schoch et al., 2004). (2) Representation of stand-scale

vegetation: macrofossil charcoal particles (Ø ≥ 2 mm) were pro-duced, deposited and buried in situ in the soil, and not trans-ported from extra-local sources (Ohlson & Tryterud, 2000). (3)Preservation in soils: soil charcoal is not only biologically andchemically inert, but also resistant to physical fragmentation,allowing in situ preservation in the soil profile over relevanttimescales (Preston & Schmidt, 2006; de Lafontaine & Asselin,2011, 2012; de Lafontaine et al., 2011). Consequently, a frac-tion of the original charcoal produced by fire persists as macro-scopic pieces suitable for wood anatomy. (4) Ubiquity in soils:macroscopic charcoal is a component of virtually any soil type.Indeed, Glinka (1914) found that there was almost no soil pro-file from Asiatic Russia in which charcoal particles did not occurin the upper horizon while Hesselman (1917) concluded that itwould be difficult to lay out a single square metre in aScandinavian forest where charcoal could not be found, andTryon (1948) suggested that charcoal is one of the constituentsfound in the soil of all forest types throughout the world. (5)Reliable direct 14C-dating: recent technical advances in accelera-tor mass spectrometry radiocarbon dating allows direct 14C-dating of small macrofossil charcoal pieces (down to 2 mg)extracted from soils. Charcoal has been acknowledged as themost reliable material for radiocarbon dating because of its highcarbon content and low risk of chemical contamination (Libby,1955). Hence, soil macrofossil charcoal analysis might be usedto infer local vegetation, climate or wildfire long-term history atthe stand scale whenever other palaeoecological approaches areunavailable or do not have the required spatial resolution (Talonet al., 2005; de Lafontaine & Payette, 2011, 2012; Ohlson et al.,2011; Payette et al., 2012).

The objective of this study is to assess whether soil macrofossilcharcoal analysis can be used to uncover glacial microrefugiahypothesized from previous genetic analyses and species-distribu-tion modelling.

Materials and Methods

Study species and biogeographical model

European beech (Fagus sylvatica L.) is one of the most abundant,widespread and economically important broadleaved tree speciesin Europe (Muhs & von Wuehlisch, 1993). As such, it has beenthe subject of studies in various fields including ecology, palaeoe-cology and genetics. Based on joint inferences from complemen-tary palaeoecological and genetic datasets, Magri et al. (2006)proposed a model for beech glacial refugia and postglacial expan-sion. According to this model, beech survived the last glacialperiod in multiple refugia including southern macrorefugia inthe Mediterranean peninsulas, as well as some northern microref-ugia in Eastern Europe, which suggests that the species has life-history traits allowing it to persist through the LPG interval atrelatively high latitudes (Bhagwat & Willis, 2008). In the greatplains of western and central Europe, extensive tracts of beechforest originated from a glacial refugium located in the area occu-pied by modern Slovenia. By contrast, beech lineages from south-ern refugia might have spread locally but were not the origin of

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poleward postglacial recolonization over most of Europe (Magriet al., 2006). Throughout the northern part of its range (north of46°N), beech grows at low elevations; in the southern part (southof 46°N), it is found mostly at altitudes above 1000 m (vonWuehlisch, 2008). At such elevations, wetter environmental con-ditions caused by orographic effects offer protection againstdrought, which is known to limit beech growth (Jump et al.,2006; Piovesan et al., 2008). In Western Europe, the southernlimit of beech distribution at low elevation (below 100 m abovesea level (asl)) is located in southwestern France in the Landes deGascogne area, between 44 and 45°N (Fig. 1, Timbal & Duc-ousso, 2010). The area of 13 000 km2 forms a wide triangleshape, bordered by the Gironde Estuary to the North, the Gar-onne River to the East, the Adour River to the south and theAtlantic Ocean to the West (Fig. 1). This low-lying area is char-acterized by a flat surface gently inclined towards the ocean. Thisunique situation along the coast allowed for the natural transportof sands far inland, yielding a thin, poorly developed, acidic cov-ersand, usually 1–2 m thick, interspaced with sporadic geologicaloutcrops from calcareous, clay and loamy Tertiary (Oligocene

and Miocene) deposits (Klingebiel & Legigan, 1985; Bertranet al., 2013). Maritime pine (Pinus pinaster) has been heavilyplanted throughout the area since the 18th century, resulting inthe development of a monospecific intensively exploited forest.Beech is virtually absent in the region except for isolated standsrestricted to a few favourable microsites that contrast with theregional matrix (Fig. 1, Table 1, Timbal & Ducousso, 2010).Indeed, the few beech stands are either gallery forests located ingorges or steep slopes along rivers and streams or characterized bypeculiar edaphic conditions (local geological outcrops) withrespect to the surrounding coversand (Table 1, Timbal & Duc-ousso, 2010). Riparian microsites could act as climatic buffersagainst unfavourable regional climate including cold and dryconditions of the LPG interval, as well as extant warm and dryconditions at the southern, low-elevation edge of beech distribu-tion. Similarly, geological outcrops could have allowed localbeech persistence within unsuitable coversand matrices since theirinception 35 000 yr BP (Bertran et al., 2009, 2011, 2013). Unfor-tunately, prospecting surveys have shown that sediments in theLandes de Gascogne containing organic deposits appropriate forpalaeoecological reconstructions are rare (Faure & Galop, 2011).Thus, the few available palaeoecological records from the area(mapped in Fig. 1) seldom extend beyond the Holocene (the last11.5 kyr) and those that do suffer from insufficient temporal res-olution.

Empirical palaeoecological information on the origin of thesemarginal populations is missing but the historical presence ofbeech in the Landes de Gascogne was hypothesized by indirectevidence. First, species-distribution models linked to estimatesof LPG interval climate indicate that climatic conditions couldhave allowed beech persistence in this area during the LPGinterval (Svenning et al., 2008). Second, beech populations fromthe Landes de Gascogne do not group together with lineagesfrom known refugia, including those of the Pyrenees, but insteadbelong to distinct genetic lineages, suggesting glacial persistencein the area rather than postglacial colonization originating froman extra-regional source (de Lafontaine et al., 2013a). Further-more, unusually high genetic differentiation among small beechpopulations within this region suggests prolonged isolation of atleast some of the beech stands in the Landes de Gascogne.Accordingly, beech could have been restricted to multiple long-term microrefugial stands in the Landes de Gascogne, each ofwhich occupied a favorable microsite surrounded by a regionalmatrix characterized by unsuitable conditions during a full gla-cial/interglacial cycle, up to the present day. During the last gla-cial period, the Landes de Gascogne area was a periglacial sanddune-dominated desert (Bertran et al., 2011, 2013). If beech wasindeed present in the region throughout the LPG intervaldespite these obviously unsuitable arid conditions, it is quite rea-sonable to hypothesise that it was restricted to some of the exactsame climatically or edaphically suitable microsites as those oftoday. Thus, using soil macrofossil charcoal analysis, our specificobjectives are to assess the presence of one or more cryptic gla-cial microrefugia for beech in the Landes de Gascogne, and touncover the glacial and postglacial history of each of the extantbeech stands.

Fig. 1 Location of sampling sites in France. The Landes de Gascogne areais shown in green. Yellow circles, location of sampled beech stands; redcircle, the toponym sample site without beech. Blue stars, location ofpollen stratigraphies (Moura (Reille, 1993); SO4; SO6 (Diot & Tastet,1995); Ang: Anguilleyrons; Hon: La Hounteyre; Bor: Bordelounque; Hub:La Hubla (Faure & Galop, 2011)). Blue asterisks, location of Aeolian duneswith paleosols from which either charcoal particles (SLM: Saint-Laurent-

M�edoc (Bertran et al., 2009); BdM: Bois-de-Marsacq (Bertran et al.,2011); Bel: Belin-B�eliet; Sab: Sabres (L. Sitzia et al., unpublished)) orpollen (CeJ: Cestas-Les Pins de Jarry (Bertran et al., 2009)) were identified.Pink octagon, Bordeaux urban area.


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Study sites

A total of 12 study sites (plot size: 509 10 m) were sampled in2011 and 2012 (Fig. 1, Table 1). Nine are either beech or beech–oak (Quercus robur, Q. pyrenaica, Q. petraea) stands, one is a mar-itime pine plantation that replaced a beech stand clear-cut in2007, and one is a managed stand containing beech mixed withindigenous and planted exotic tree species (e.g. Cedrus atlantica,Quercus rubra, Pseudotsuga menziesii, Thuja occidentalis)

(Table 1). To our knowledge, these 11 stands represent all theextant beech occurrences in the Landes de Gascogne (Timbal &Ducousso, 2010). Another site included in the study is an oak-dominated stand where beech is currently absent (Table 1). Itwas included because it constitutes a good candidate in which toassess whether beech was more widespread in the area beforeincreased human activities, as suggested by a recent floristicmodel (E Silva et al., 2012). As with the 11 extant beech stands,this site contrasts with the regional matrix: it is a riparian gallery

Table 1 Location and details of study sites

Abbra Site nameLatb

(°N)Longc

(°W)Altd

(m a.s.l.) Extant vegetation

Soile

Topography

Putative in situ

features decoupledfrom regionalenvironmentType Texture

AGE Masd’Agenais

44.39 0.19 50 Beech-oak forest stand withhornbeam

Cambisol Loam Mild slope (smallstream valley)

HydricThermalEdaphic

CAZ Cazats 44.48 �0.18 80 Riparian gallery forest.Beech-oak forest standwith hornbeam

Cambisol Loam Steep slope(small streamvalley)


CIRA Ciron A 44.40 �0.33 60 Riparian gallery forest. Old-growth beech dominatedstand with linden andhornbeam

Cambisol(calcic)

Sandyloam

Gorge (alongCiron River)


CIRB Ciron B 44.39 �0.31 60 Riparian gallery forest.Old-growth beechdominated stand withlinden and hornbeam

Cambisol(calcic)

Sandyloam



CIRC Ciron C 44.38 �0.28 60 Riparian gallery forest.Old-growth beechdominated stand withlinden and hornbeam

Cambisol(calcic)

Sandyloam



CUR Curton 44.48 �0.58 80 Maritime pine plantationreplacing a beech forestclear-cut in 2007

Cambisol(gleyic)

Clay Flat terrain Edaphic

ESC Escource 44.17 �1.04 60 Riparian gallery forest.Beech individuals of allages within oak-dominated stand

Podzolicarenosol

Sand Steep slope(small streamvalley)

HydricThermal

LAV Laveyron 43.91 �0.22 90 Beech-oak forest stand withhornbeam

Cambisol Loam Mild slope (smallstream valley)


ROQ Roquefort 44.03 �0.33 70 Riparian gallery forest.Old-growth beechdominated stand withlinden and hornbeam

Podzolicarenosol

Sand Gorge (alongDouze River)

HydricThermal

SYM St-Symphorien 44.44 �0.47 60 Mixed managed forestcontaining exotic species

Podzolicarenosol

Sand Flat plateaualong HureRiver banks

Hydric

VIL Villenave 43.95 �0.79 60 Riparian gallery forest.Beech-oak forest standwith alder and elder

Podzolicarenosol

Sand Flat plateau,along Bez Riverbanks

Hydric

HAOU Haou = beechtoponym

44.38 �0.77 50 Riparian gallery forest.Oak-dominated standwith birch. Beech is absent.

Podzolicarenosol

Sand Gorge (alongEyre Rivertributary)

HydricThermal

aSite name abbreviation.bLatitude.cLongitude.dAltitude.eSoil classification follows IUSS Working group WRB (2006).



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forest located in a protected gorge (Table 1). Furthermore, it islocated c. 2 km next to a beech toponym (Haou, a place namederived from hetre, which means beech in French), which couldsuggest local beech presence in the recent past (Tarze et al.,2009).

Charcoal extraction and taxonomic identification

Soil macrofossil charcoal analysis was based on the sampling of25 surficial mineral soil cores at each site. Cores were sampledevery 5 m along the plot perimeter with a core in the middle ofthe plot. After removing the thin organic horizon, a 15 cm longmineral soil core (750 cm3) was extracted with a root auger(SDEC, Reignac-sur-Indre, France). Each sample was immersedfor 12–24 h in a dispersing solution of sodium hydroxide(NaOH – 1%) or sodium hexametaphospate ((NaPO3)6 – 2.5%)for sand and loam/clay soils, respectively. The material was thenwashed under running water for 10 min in sieves with mesh sizesof 4 and 2 mm using a vibratory wet sieve shaker (Retsch, Haan,Germany). These first steps also provided an initial cleaning ofcharcoal surface from contamination by organic matter. Charcoalwas then extracted from the mineral fraction by floatation andmanual sorting under a binocular microscope (Olympus, Tokyo,Japan). At this stage, any visible remaining charcoal contamina-tion (e.g. rootlets, hyphae) was removed manually. Charcoalparticles were dried in a stove (Heraeus, Hanau, Germany) at60°C for 8 h and weighed to the nearest 0.1 mg. They were iden-tified to the lowest taxonomic rank possible under an opticalmicroscope (magnification: 9100, 9200, 9500) mounted withepiscopic light source (Olympus) using a charred wood referencecollection from regional species (Anthracoth�eque de Bordeaux,UMR BIOGECO, INRA, France; de Lafontaine et al., 2013b)and botanical keys (Vernet et al., 2001; Schoch et al., 2004). Atsites where many charcoal particles were found (AGE, CUR andLAV), taxonomic identification was limited to a random subset(Supporting Information Table S1). During botanical identifica-tion, potential organic contamination was rigorously assessed atup to 9500 magnification.

Radiocarbon dates

A total of 140 charcoal particles were radiocarbon dated by theAccelerator Mass Spectrometry technique at the Pozna�n Radio-carbon Laboratory, Poland, after standard acid-alkali-acid pre-treatments necessary to remove humic and fulvic acids, as well asadhering secondary carbonates (Alon et al., 2002). The finalnumber of dates obtained for each site was the outcome of aniterative process. At each site, at least five particles were initiallydated (except for toponym site HAOU, n = 4 dates). After apreliminary analysis based on the results of this initial sampling,further dates were obtained from sites where the accumulationcurve suggested that fire events were likely to have been missed(see later). This step was then repeated until the total of 140 sam-ples was reached. Selection of charcoal fragments to be dated wasbased on dry weight of the particles (> 2.5 mg; lower limit forradiocarbon dating), botanical identification (focusing on beech

but including a few other species), spatial distribution of the 25soil cores within the plot, and randomness (among the particlesthat fitted the above criteria). Radiocarbon dates were calibrated(cal yr before present (BP), 2-sigma) using calibration dataset Int-Cal13.14c (Reimer et al., 2013) implemented in CALIB 7.0(Stuiver & Reimer, 1993; Stuiver et al., 2005).

Reconstruction of stand-scale histories

At each site, the determination of fire events was based on cumu-lative probability analysis (Meyer et al., 1992) using the ‘SumProbabilities’ option in CALIB 7.0 (Stuiver et al., 2005) to plotthe probability that a given event occurred at a particular time(Fesenmyer & Christensen, 2010; de Lafontaine & Payette,2011, 2012). This method sums the probability distributions ofall calibrated dates and therefore takes into account the uncer-tainties inherent in radiocarbon dating. The radiocarbon age of acharcoal fragment corresponds to the time when the wood thatyielded charcoal was produced and not to the actual age of a fireevent. This ‘inbuilt-age’ error is in addition to the radiometricerror and implies that the radiocarbon age can be older than theactual date of the fire which produced charcoal (McFadgen,1982; Gavin, 2001). The inbuilt-age error depends on stand agestructure and rate of wood decay, and it may be significantenough to require addressing in sites with long-lived trees and/ordecay-resistant wood, as in coastal forests of western USA andCanada, where trees may attain ages of c. 1000 yr (Gavin, 2001).However, in sites with short-lived trees and fast decaying wood,the radiocarbon dates of charcoal approximate the actual dates offire (Filion, 1984; de Lafontaine & Payette, 2011, 2012). In thisstudy, correction for inbuilt age error was deemed unnecessarybecause the oldest trees in beech stands of the Landes de Gasco-gne are younger than 200 yr (E Silva, 2010), and the rate of wooddecay is short – generally c. 30 yr in beech stands throughout therange (Koop, 1981; Kraigher et al., 2002; von Oheimb et al.,2007; M€uller-Using & Bartsch, 2009). Decay should be evenfaster in warmer stands at the southern limit of the beech range(Mackensen et al., 2003).

Charcoal particles were originally buried in the mineral soilfraction by tree fall and uprooting or other disturbances affectingsoil stability (Gavin, 2003). Their position in the soil profile wasfurther reworked by subsequent taphonomical processes (e.g. fur-ther uprooting events, freeze–thaw cycles, biotic activity and sur-face erosion), producing mixed soil horizons up to 1 m deep(Lutz, 1940; Brown & Martel, 1981; Bormann et al., 1995;Gavin, 2003; Preston & Schmidt, 2006). As a consequence, theyare not stratified in soils (Carcaillet, 2001; Gavin, 2003; Fesen-myer & Christensen, 2010) except in dune environments andloess sequences where they are distributed in superposed organiclayers (Filion, 1984; Haesaerts et al., 2010). It is thus importantto assess whether most fires have been detected given our sam-pling effort because our capacity to reconstruct vegetation historyfrom fossil charcoal depends on the number of past fire eventsthat produced these charcoal particles. For this purpose, we com-puted asymptotic accumulation curves from the 14C data to esti-mate by extrapolation the expected maximum number of fire





events that occurred at each site (extrapolation procedure detailedin Methods S1; de Lafontaine & Payette, 2011, 2012). Keepingin mind that it was not the goal of this study to reconstruct thelong-term fire history, the extrapolation procedure should beinterpreted as a relative reflection of the completeness of our fossilrecord, not as an indication of the actual number of fire events.At each stand, beech history was evaluated by assessing presenceor absence of at least one beech charcoal particle during eachrecorded fire event.

Reconstruction of regional history

A regional fire history of the Landes de Gascogne was inferred byrunning the cumulative probabilities analysis on the pooledradiocarbon dataset from all 11 beech stands (excluding toponymsite HAOU). Vegetation history of the beech stands in the Lan-des de Gascogne was inferred from taxonomically identified andradiocarbon-dated charcoal particles of all beech stands. Thenumber of charcoal particles of each 14C-dated tree taxon wascounted during each successive 500 cal yr BP interval to recon-struct past forest composition through time (we accounted forthe fact that the 2r calibrated range of the age of each charcoalparticle could span more than one 500 yr interval by using deci-mal values, weighting each charcoal particle by its probability ofoccurrence during the corresponding time interval). The regionalhistory was tentatively interpreted in the context of publishedpalaeoecological evidence. However, regional terrestrial palaeo-ecological data are scant because suitable depositional environ-ments are scarce (Fig. 1) and chronologies are often incomplete.South of 46°N, the nearest pollen records extending into thePleistocene come from sites in the Pyrenees, Massif Central orthe Alps (e.g. de Beaulieu & Reille, 1984; Reille & de Beaulieu,1990; Reille & Andrieu, 1995). In the context of our study, theserecords are inappropriate to help interpreting our regional recon-struction based on local-scale lowland data. Instead, we rely oninferences from the North Atlantic marine records (includingpalaeoclimatic, palaeovegetation and palaeofire reconstructions).Although these are not spatially explicit, they provide a detailedchronology (at the century to millennial scale) of the westernEuropean vegetation and wildfire regimes in direct relation withclimatic events of the North Atlantic (Combourieu Nebout et al.,2002; Sanchez Go~ni et al., 2002, 2008).

Results

Charcoal abundance and taxonomic identification

Charcoal particles were found in the soil of all sampled sites(n = 19 to 978 particles per site, total n = 3187 particles, Fig. 2,Table S1). The botanical composition of the charcoal recordrelied on 1232 successful taxonomic identifications, representing39% of the complete record (Table S1). Macrofossil charcoal ofbeech was found at all 11 extant beech stands but not at the topo-nym site (Fig. 2). A total of 24 taxa were identified. Beech, decid-uous oaks and maritime pine were found with the highest relativeabundances (representing 34%, 20% and 20% of the complete

record, respectively) (Fig. 2, Fig. S1). These, as well as other taxafound more sporadically in the macrofossil charcoal record (e.g.Acer campestre, Alnus sp., Betula sp., Carpinus betulus, Castaneasativa, Corylus avellana, Ilex aquifolium, Fraxinus sp., Populus sp.,Salix sp., Tilia sp.), are current components of the Atlantic decid-uous forest of Europe (Ozenda, 1982; Polunin & Walters,1985). A noteworthy exception is charcoal of Scots pine (Pinussylvestris), a cold-tolerant forest species currently absent in thearea, which was found at five of the 12 sites (Fig. 2; note thatcharcoal of the Pinus type sylvestris section includes Pinussylvestris, P. nigra and P. mugo, but in the current study, they areassumed to refer to P. sylvestris, the only pine species inventoriedfor the Lateglacial within the study region; Bertran et al., 2009,2011).

Stand-scale histories

At each site, between one and at least 14 fire events weredetected. Fig. 3 illustrates the stand-scale records along with theaccumulation curves used to estimate stand-scale fire history byextrapolation. Parameters of the accumulation curves are shownin Table S1, while Table S2 reports the 140 AMS radiocarbondates. The comprehensive analysis of stand-scale histories is avail-able in Notes S1.

Regional history

The regional fire history of beech stands extends from beyond thelimit of radiocarbon dating (at 50 000 cal yr BP; Libby, 1955;Bronk Ramsey et al., 2012) up to the present-day and displays amarked shift in fire activity at c. 10 000 cal yr BP (Fig. 4a).Between >51 000 and 10 000 cal yr BP, a period of very low fireactivity characterized by few, sporadic fire events interspaced byirregular intervals with no evidence of fire was identified (Fig. 4a).

Fig. 2 Soil charcoal record at each sample site. Each bar represents thetotal number of charcoal particles indicating the number of charcoalparticles successfully identified (black), the number of charcoal particlesthat were assayed but not successfully identified (dark grey) and thenumber of charcoal particles that were not assayed (light grey). Theasterisk is a reminder that the corresponding study site is the toponym sitewithout extant beech population. Pie-charts express the relativeabundances of taxonomic identifications (green, Fagus sylvatica; yellow,Quercus sp.; red, Pinus pinaster; blue, Pinus type sylvestris; grey, othertaxa).



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By contrast, beech stands from the Landes de Gascogne were sub-jected to an uninterrupted period of elevated fire activity since10 000 cal yr BP (Fig. 4a). At the regional scale, beech charcoalwas recovered from every interval in which fire was recorded

(compare Fig. 4a with b). Specifically, the charcoal record consti-tutes direct fossil evidence that beech was present in the area, atleast intermittently, during the following intervals: before 51 000,between 43 300 and 40 000, between 32 900 and 31 300,

(a) (b)

(c) (d)

(e) (f)

(g) (h)

(i) (j)

(k) (l)

(m) (n)

(o) (p)

(q) (r)

(s) (t)

(u)(v)

Fig. 3 Stand-scale fire and vegetationhistories of beech stands from the Landes deGascogne. Left panels, cumulated probabilityof the calibrated 14C dates. ‘+’, differentcalibrated 14C dates (i.e. different fire events:green crosses, a fire event for which at leastone piece of beech charcoal was recovered;yellow crosses, no beech charcoal wasrecovered).’?’, a radiocarbon date > 51 00014C yr before present (BP). Right panels,accumulation curves of the number of fireevents recorded based on calibrated 14Cdated charcoal particles. Blue polygons, 95%confidence interval envelopes of 100randomly permuted accumulation curves.Boxplots of the 100 permutations indicatelower quartile, median and upper quartile;whiskers length are 1.59 interquartileranges. Black lines, extrapolation curvesfitted from each mean accumulation curveusing an asymptotic, negative exponentialfunction.





between 15 900 and 14 800 and between 13 000 and 12 700 calyr BP; as well as continuously since 9000 cal yr BP (Fig. 4b). Onlythe cold-tolerant species Pinus sylvestris was found along withbeech during the Pleistocene. Scots pine charcoal was consistentlypresent in the early Holocene between 10 000 and 8000 cal yr BP

but only one occurrence is recorded subsequently, at 4800 cal yrBP (Fig. 4b). Deciduous oaks were found continuously since5600 cal yr BP (Fig. 4b). Pinus pinaster was only found since thelast millennium with a notable increase during the last 500 yr(Fig. 4b). Other dated taxa (Alnus sp., Betula sp., Ilex aquifolium,Castanea sativa) were found since 9200 cal yr BP (Fig. 4b).

Discussion

We found macrofossil charcoal particles buried in the soil of allsampled sites, implying that our study was not limited by char-coal availability. Our radiocarbon dataset extended before 51 00014C BP, that is beyond the intrinsic limit of radiocarbon method-ology (Libby, 1955; Bronk Ramsey et al., 2012). This supportsthe assertions about ubiquity and long-term preservation of macro-fossil charcoal in soils from natural ecosystems (de Lafontaine &Asselin, 2011, 2012). Previous studies have shown that the analy-sis of soil macrofossils provides robust estimates of the minimumnumber of past fire events, as well as supplying direct fossil

evidence for the historical presence of taxa at the stand scale(Talon et al., 2005; de Lafontaine & Payette, 2011, 2012; Payetteet al., 2012). Yet, palaeoecological approaches relying on soilcharcoal remain underutilized (Willis & van Andel, 2004).Unlike most other palaeoecological methods, soil macrofossilcharcoal records do not yield continuous stratigraphic sequences(Carcaillet, 2001; Fesenmyer & Christensen, 2010). Instead, theyprovide ‘snapshots’, at different time frames, of the local vegeta-tion that was burnt during fire events (Willis & van Andel,2004).

In our study, the soil of each stand was thoroughly searchedfor charcoal particles using systematic sampling with high spatialresolution. Using this sampling scheme, we found beech macro-fossil charcoal in all extant beech stands in the Landes de Gasco-gne, whereas the only site where no beech macrofossil was foundis also the only site currently devoid of beech. Except for cold-tolerant Scots pine, which is currently absent in the area,taxonomic identifications of our charcoal record indicated com-ponent taxa of the extant Atlantic deciduous forest of Europe(Ozenda, 1982; Polunin & Walters, 1985).

The fire history inferred by pooling together radiocarbonrecords from the 11 beech stands outlined a drastic change of thefire regime at the onset of the Holocene. A period of low fireactivity with few, sporadic fire events interspaced by irregular

(a)

(b) Fig. 4 Regional fire history (a) andradiocarbon dated macrofossil charcoalassemblage (b) of the beech stands in theLandes de Gascogne. ‘?’, a radiocarbon date> 51 000 14C yr before present (BP). (a)Histograms represent the cumulatedprobability of the calibrated 14C dates. Thehorizontal bar above graph illustratesintervals for which beech (green) or otherspecies (grey) were detected in the charcoalrecord, as well as intervals lacking charcoalrecord (white). (b) Number of charcoalparticles of Fagus sylvatica (green),Quercus

sp. (yellow), Pinus pinaster (red), Pinus typesylvestris (blue) and other taxa (Ilexaquifolium, Betula sp., Castanea sativa andAlnus sp., grey) recorded in each 500 cal yr BP

interval, respectively. Numbers (1–8) depictindividual charcoal particles found in thePleistocene, all of which fall into more thanone 500-yr bin.



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intervals lacking evidence of fire was identified during the last gla-cial period, whereas the area was submitted to an uninterruptedperiod of high fire activity during the Holocene. In principle,processes other than fire history could have influenced the macro-fossil charcoal record over the past 51 000 yr. Specifically, char-coal particles originating from older fire events might beunderrepresented as a consequence of soil erosion (Jaff�e et al.,2013) and perhaps local deflation. This could have exaggeratedthe shift observed between the sparse charcoal record of the lastglacial and the prolific record during the Holocene. However, theassumption that the regional radiocarbon record actually reflectsthe fire history is consistent with the literature. Indeed, palaeocli-matic reconstructions based on high resolution multiproxy analy-ses of deep-sea records have suggested that episodes of low fireactivity in Western Europe were closely associated with periodsof drought, while fire activity increased during wetter intervals(Daniau et al., 2007, 2009). This pattern has been explained byfuel availability: during periods of drought associated with coldintervals (e.g. during the LPG), low arboreal biomass in WesternEurope would explain the low fire activity, whereas the warmerand wetter periods (e.g. during the Holocene) characterized byincreased fuel availability would result in higher fire activity (Da-niau et al., 2007, 2009). Our reconstruction of the Landes deGascogne fire history could indeed reflect the overall biomassavailability in the regional matrix, with low woody fuel in theperiglacial desert during the LPG interval and increased fuel loadsince the afforestation of the area at the beginning of the Holo-cene (Bertran et al., 2011, 2013). Most beech stands in the Lan-des de Gascogne are gallery forests located in protected gorges orslopes along rivers or streams. Typically, such microhabitats arenot fire-prone but instead constitute natural firebreaks (Everettet al., 2003; Pettit & Naiman, 2007; cf. also the greaterabundance of macrofossils found in open areas such as CUR thanin river gorges). Yet, we found charcoal in all these sites. We thusassume that most wildfire events recorded in these protected siteswere ignited ex situ during intervals of overall increased arborealbiomass availability. Hence, keeping in mind that the absence ofcharcoal in the record is no proof for the absence of vegetation atthe stand scale, it is conceivable that trees survived in thesefavourable microsites even at times when the regional forest coverwas low.

Regarding the origin of beech in the Landes de Gascogne,three alternative scenarios were conceivable before any analysis.The first scenario (glacial absence) implies that beech was com-pletely absent in the area during the last glacial period and recol-onized the Landes de Gascogne during the Holocene. Magri et al.(2006) suggested that beech expanded in regions north of the Py-renees between 4500 and 3200 cal yr BP. However, the Landes deGascogne were absent from their broadscale biogeographicalreconstruction. In the published terrestrial palaeoecologicalrecords from the area (Fig. 1), there was no evidence of beechbefore the Holocene (Fig. 5). Although most published palaeo-ecological records lack appropriate temporal resolution, there is acommon historical pattern across the published data: Scots pinewas present during the late Pleistocene and early to middle Holo-cene until c. 4000 cal yr BP, beech pollen was found during the

middle to late Holocene, and maritime pine appeared in pollenrecords during the last millennium (Fig. 5). Our regional Holo-cene reconstruction for these three species parallels that of the lit-erature, suggesting that our palaeoecological approach wasreliable at this scale. More importantly, we extend back therecord for both beech and Scots pine. The low pollen percentage(c. 10%) of Scots pine found in dune palaeosols dating back tothe last glacial period (26 500–28 500 cal yr BP) was interpretedas originating from long-distance (extra-regional) dispersal (Ber-tran et al., 2009; Fig. 5). We found macrofossils of charred Scotspine at some sites during the last glacial period, suggesting thatthe low pollen percentage found by Bertran et al. (2009) could aswell originate from a local source (Bennett, 1984; Cheddadiet al., 2006). Finally, thanks to our local-scale approach focusedon beech stands, we found direct evidence of the continuous pres-ence of beech in the Landes de Gascogne since the early Holo-cene, as well as sporadically during the Pleistocene, allowing us toformally rule out the scenario of continuous glacial absence.

The second scenario (millennial-scale dynamics) implies thatbeech alternated between regional presence and absence duringthe Pleistocene while tracking rapid climatic variability followingthe Dansgaard–Oeschger oscillation and Heinrich stadials (Gentyet al., 2005b; Overpeck & Cole, 2006). High-resolution pollenanalyses of marine sediment cores have documented the sensitiveresponse of Southwestern European vegetation to rapid climatevariability during the last glacial period, with rapid forest devel-opment during Dansgaard–Oeschger interstadials and forest con-traction during stadials and Heinrich events (CombourieuNebout et al., 2002; Sanchez Go~ni et al., 2002; Naughton et al.,2007; Fletcher & Sanchez Go~ni, 2008; Fletcher et al., 2010).However, Sanchez Go~ni et al. (2008) have shown that the ampli-tude of Mediterranean and Atlantic forest expansions differswidely among interstadials and that afforestation of the mid-Atlantic region corresponded to strong climatic warming ofDansgaard-Oeschger interstadials 12 and 14 (Sanchez Go~niet al., 2008; Fletcher et al., 2010). Although we cannot rigorouslyexclude millennial-scale beech dynamics from our discontinuousdataset, the glacial intervals when beech was present in our radio-carbon record (interstadials 10–11 and 5–6, and Heinrich sta-dial-1; Fig. 5) do not match with intervals of major Atlanticdeciduous forest expansion over Southwestern Europe (SanchezGo~ni et al., 2008; Fletcher et al., 2010). This implies that beechpresence in the study region during the last glacial period was notstrictly a consequence of rapid expansion of Atlantic flora asbeech was also found during other, colder periods.

The third scenario (glacial microrefugia) implies that beech wascontinuously present at some favorable microsites throughout theLPG interval. We found no direct evidence of beech in our mac-rofossil charcoal dataset during the Last Glacial Maximum(LGM; between 26.5 and 19 kyr BP, Clark et al., 2009). How-ever, beech was detected during an even harsher climatic episode(Heinrich stadial-1), suggesting that it could have persistedthroughout the LGM. LGM refers to the most recent periodwhen global ice sheets reached their maximal volume and shouldnot be viewed simply as a climatic extreme everywhere (McM-anus et al., 2004; Clark et al., 2009; Tzedakis et al., 2013).





Indeed, palaeoclimatic data and models suggest that Heinrich sta-dial-1 (between c. 19 and 14.6 kyr; equivalent to the OldestDryas of northwest Europe; Sanchez Go~ni & Harrison, 2010;Stanford et al., 2011; Clark et al., 2012; Caulle et al., 2013) wascolder and dryer than the LGM in Western Europe (McManuset al., 2004; Genty et al., 2005a; Kageyama et al., 2005; Fletcher& Sanchez Go~ni, 2008; Morell�on et al., 2009; Moreno et al.,2010; Sanchez Go~ni & Harrison, 2010; Stanford et al., 2011;Clark et al., 2012; Caulle et al., 2013). Specifically, in the Landesde Gascogne, model–data comparison has indicated that Hein-rich stadial-1 was c. 1°C colder and c. 20% drier than the LGM(Kageyama et al., 2005). Interestingly, charcoal particles fromPinus type sylvestris and Fagus sylvatica (16 784–16 239 and15 839–14 786 cal yr BP, respectively) are completely includedwithin this interval, implying that beech and Scots pine were

present in the area during the coldest and driest interval of theLPG (Fig. 5). Given that Heinrich stadial-1 immediately fol-lowed the LGM, it would be surprising if these species wereabsent during the LGM and recolonized the area during harsherclimatic conditions of Heinrich stadial-1. This lends support tothe existence of at least one microrefugium for these species.Thus, the scenario of continuous regional presence in glacial mi-crorefugia cannot be discarded. In fact, it is the only scenario thataccounts for the distinct genetic structure of beech stands identi-fied in the region (de Lafontaine et al., 2013a).

It must be acknowledged that our radiocarbon dataset focusedprimarily on beech. To test whether beech arrived earlier than theoaks while accounting for sampling bias, we used a resamplingprocedure (Notes S2) that suggests that beech was present earlierthan oaks within the sampled stands, a result lending support to

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

(k)

(l)

(m)

Fig. 5 Multiproxy terrestrial palaeoecological record of Fagus sylvatica, Pinus pinaster and Pinus type sylvestris in the Landes de Gascogne over the last50 000 yr compared with Greenland temperature variations. (a) Regional fire history of the beech stands. (b) Northern Greenland ice core (NGRIP) d18Odata (NGRIP members, 2004). Light shaded areas in the background, glacial stadials; dark grey areas numbered H1–H5, correspond to major cooling(Heinrich) stadials (Bond & Lotti, 1995); unshaded areas, the Holocene and Dansgaard-Oeschger warmer interstadials (marked i1–i12). (c–m) Each boxrepresents a terrestrial palaeoecological record. The length of each box corresponds to the total duration of the radiocarbon dated record. Vertical blacklines inside a box represent radiocarbon dates of stratigraphic levels within pollen cores. Green, red and blue colours, records of Fagus sylvatica, Pinuspinaster and Pinus type sylvestris, respectively. ‘?’ together with fading colours, an absence of a suitable chronology, thus preventing accurate dating ofthe appearance or disappearance of the corresponding taxa in the paleoecological record. (c) Palaeoecological record from pollen (Cestas-Les pins de Jarry(Bertran et al., 2009)) or charcoal (marked char) (Saint-Laurent-M�edoc (Bertran et al., 2009); Bois-de-Marsacq (Bertran et al., 2011); Belin-B�eliet andSabres (L. Sitzia et al., unpublished)) buried in paleosols from Aeolian dunes. (d) Le Moura: pollen from lacustrine sediments core (Reille, 1993). (e)Bordelounque: pollen from peatland core (Faure & Galop, 2011). (f) SO4: pollen from marsh sediments core (Diot & Tastet, 1995). (g) La Hubla: pollenfrom peatland core (Faure & Galop, 2011). (h) SO6: pollen from marsh sediments core (Diot & Tastet, 1995). (i) La Honteyre: pollen from peatland core(Faure & Galop, 2011). (j) Charcoal record (2-sigma calibrated range) of toponym site HAOU. (k) Anguilleyrons: pollen from peatland core (Faure &Galop, 2011). (l) Composite charcoal record (2-sigma calibrated range) of all beech stands. (m) Composite multiproxy terrestrial record pooling informationfrom (c) to (l).



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the hypothesis that beech endured glacial conditions at higher lat-itudes than oaks (Bhagwat & Willis, 2008). Regarding maritimepine, it was found exclusively in the last millennium and wasmost abundant during the last 500 cal yr BP, a pattern alsoreported in published regional pollen diagrams (Fig. 5). Thecharcoal and pollen records of maritime pine thus appear tightlyassociated with human activity and likely reflect extensive planta-tions of this species in the area since the 18th century.

If the microrefugia scenario holds, then the question of whichstudy sites were beech refugia arises. First, the three sites withbeech charcoal dated from the Pleistocene represent the best can-didates and thus merit deeper investigations. Second, at two fur-ther sites beech charcoal was found that dated to the Holocenebut older fire events were recorded from charred wood particlesof Scots pine, implying that the sites were likely to have been atleast partially forested during the last glacial period. Furtherresearch at these sites could perhaps reveal additional beech mi-crorefugia. Third, five other sites lack a charcoal record before themiddle or late Holocene. From our charcoal data alone, it is thusimpossible to disentangle glacial persistence from postglacialrecolonization at these sites. Last, at one site, there was a singlerecent fire (c. 200 yr ago) in which beech was found along withmaritime pine. In view of the presence of exotic species in thismanaged stand, we conclude that the extant beech populationhad been planted. Finally, we found no evidence of beech pres-ence in the only stand lacking beech but clearly further data areneeded from additional potentially suitable microsites to assessthe former extent of beech forests in the region.

The very few extant beech stands in the Landes de Gascognerepresent the southern limit of the species distribution at lowelevation. There are increasing concerns about the persistenceof such rear-edge populations in the face of ongoing globalwarming (Hampe & Petit, 2005). These beech stands arerestricted to microsites with favorable climatic or edaphic con-ditions decoupled from the surrounding landscape. Our datasuggest that this decoupling is of great antiquity and that someof these beech stands constitute long-term stable microrefugiathat have persisted through a full glacial/interglacial interval upto the present day at the same exact location, including attimes when the regional environment was largely unsuitable, asat present. Further research monitoring fine-scale climatic vari-ability of these stands and of the surrounding matrix will helpdetermine whether beech could persist in the face of climatechange or whether it is bound to disappear (Ashcroft, 2010;Dobrowski, 2011; Ashcroft et al., 2012; Keppel & Wardell-Johnson, 2012; Keppel et al., 2012).

Species distribution models represent one of the most impor-tant tools to predict the impact of global warming on the biota(Nogu�es-Bravo et al., 2008). Recently, Maiorano et al. (2013)have shown that building models with paleoecological dataensures a far better representation of species ecology (i.e.improved estimation of the fundamental niche). This is impor-tant because small change in the estimation of the niche spacebrought by the addition of palaeoecological data may have pro-found consequences on the projected new distribution of species.Saltr�e et al. (2013) compared model performance between species

distribution models based only on macrorefugia and species dis-tribution models based on both macro- and putative microrefu-gia and found that the latter had much better agreement with theobserved postglacial colonization dynamics through more accu-rate simulation of migration rate. Thus, in order to effectivelyforecast effects of climate change, it appears necessary to acquirebetter knowledge of the location of refugia (including small, dis-junct microrefugia) using robust local fossil data. In this respect,soil macrofossil charcoal analysis represents a promising avenue.

Conclusions

This study is amongst the first direct attempts at decrypting crypticforest refugia – explicitly testing the existence of glacial microrefu-gia using fossil evidence. Specifically, using soil macrofossil char-coal analysis allowed us to reconstruct the long-term history ofthe beech stands in the Landes de Gascogne area. The approachprovided historical records regarding the wildfire regime and for-est composition at the stand scale over the past 50 000 yr includ-ing during some of the coldest episodes of the last ice age.Thanks to the ubiquity and long-term preservation of soil char-coal, palaeoecological reconstructions were possible despite thelack of suitable depositional environments for pollen or macro-fossil analyses. Integrating stand-scale soil macrofossil charcoaldata to the broader context inferred from regional marine palaeo-records helped improve our understanding of the ecological his-tory of the studied stands. Thus, soil macrofossil charcoal analysisemerges as a valuable complementary approach in multiproxystudies aiming at unambiguously assessing the exact locations ofmicrorefugia.

Acknowledgements

We thank P. Bertran, A. Hampe, D. Magri, M.F. Sanchez Go~niand L. Sitzia for helpful comments on earlier versions of the man-uscript. Funding was provided by the LinkTree project (ANR BI-ODIVERSA) to RJP and INRA-EFPA ‘Projets Innovants’ 2011to G.deL. C.A.A.G. was supported by Erasmus Mundus BAPEproject fellowship and LabEx COTE project. G.deL. benefitedfrom a FQRNT postdoctoral fellowship.

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Supporting Information

Additional supporting information may be found in the onlineversion of this article.

Fig. S1 Abundance of each identified taxa in the macrofossilcharcoal record of the Landes de Gascogne area.

Table S1 The charcoal record and parameters of the accumula-tion curves used to estimate stand-scale fire history



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Table S2 The 140 accelerator mass spectrometry radiocarbondates

Methods S1 Procedure used to estimate by extrapolation theexpected maximum number of fire events that occurred at eachsite using asymptotic accumulation curves from 14C data.

Notes S1Detailed analysis of stand-scale histories.

Notes S2 Resampling procedure used to assess whether beechwas present in the Landes de Gascogne earlier than the oaks whileaccounting for a sampling bias.

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