Protected areas of Spain preserve the neutral genetic diversity of Quercus ilex L. irrespective of glacial refugia

ORIGINAL ARTICLE

Protected areas of Spain preserve the neutral genetic diversityof Quercus ilex L. irrespective of glacial refugia

Beatriz Guzmán1& Carlos M. Rodríguez López2 & Alan Forrest3 & Emilio Cano1 &

Pablo Vargas1

Received: 11 December 2014 /Revised: 27 September 2015 /Accepted: 15 October 2015 /Published online: 3 November 2015# Springer-Verlag Berlin Heidelberg 2015

Abstract Quercus ilex L. (holm oak) is a wind-pollinated,sclerophyllous tree that copes with the environmental variabil-ity of the Mediterranean climate and that displays flexibleecophysiological adaptability in relation to hydric and thermicstresses. The holm oak dominates Mediterranean woodlandson both acidic and calcareous soils and has been exposed tomanagement (dehesas) for thousands of years. Both protectedareas and glacial refugia are supposed to preserve a substantialfraction of the genetic diversity of Iberian species. Geneticdiversity was examined for 68 populations sampled through-out Spain using ptDNA SNPs, ptDNA microsatellites, andprimarily nuclear AFLPs. Protected populations did not sig-nificantly differ from nonprotected populations by any of themeasures of levels of genetic diversity. The three-level hierar-chical AMOVA indicated that a low number of protected pop-ulations harbor most of the species’ genetic diversity. In addi-tion, we found no evidence from either ptDNA or AFLP var-iation to support that populations from putative glacial refugia

are divergent genetic groups as expected during isolation.Outcrossing, anemophilous long-distance pollen dispersal,acorn transport by animals, tree reliance, and habitat availabil-ity in Spain probably played a primarily role in homogenizingallele frequency among populations. This result leads us tosuggest that extensive gene flow has been prevalent acrossSpanish populations. We conclude that glacial refugia havenot been essential to maintain the neutral genetic makeup ofQ. ilex. Nevertheless, conservation of the holm oak inprotected areas ensures protection of the species’ genetic di-versity, the most widespread woodland ecosystem in Iberiaand indirectly the four iconic, endangered animal species(black stork, cinereous vulture, Iberian lynx, western imperialeagle).

Keywords Conservation planning . Genetic conservation .

Holm oak . National park . Nonprotected areas

Introduction

The holm oak (Quercus ilex L., Fagaceae) is one of the mostcharacteristic trees of the Mediterranean climate, with popu-lations throughout the Iberian Peninsula, especially in Spain(Barbero et al. 1992), as well as extensively across theMediterranean floristic region (North Africa, Mediterraneanislands, and southern continental Europe). The holm oak is asclerophyllous tree (i.e., small, leathery, and dark leaves cov-ered with thick cuticles and small, thick-walled cells that helpto resist low water availability during summer; Read andSanson 2003) that copes with environmental variation bymeans of a flexible ecophysiological adaptability in relationto hydric and thermic stresses (Gimeno et al. 2009). It is amonoecious, wind-pollinated tree that thrives on a variety ofsubstrates and exhibits a broad morphological variation that

This article is part of the Topical Collection on Germplasm Diversity

Communicated by A. Kremer

Electronic supplementary material The online version of this article(doi:10.1007/s11295-015-0950-2) contains supplementary material,which is available to authorized users.

* Beatriz Guzmá[email protected]

1 Real Jardín Botánico, CSIC, Plaza de Murillo 2,28014 Madrid, Spain

2 Plant Research Centre, School of Agriculture, Food and Wine,Faculty of Sciences, University of Adelaide, Waite Campus, PMB1,Glen Osmond, SA 5064, Australia

3 Centre for Middle Eastern Plants, Royal Botanic Garden Edinburgh,20a Inverleith Row, Edinburgh EH3 5LR, UK

Tree Genetics & Genomes (2015) 11: 124DOI 10.1007/s11295-015-0950-2

http://dx.doi.org/10.1007/s11295-015-0950-2

http://crossmark.crossref.org/dialog/?doi=10.1007/s11295-015-0950-2&domain=pdf

has resulted in the recognition of many subspecies and varie-ties (Albert and Jahandiez 1908; do Amaral 1990).Adaptability to the Mediterranean climate is manifested bythe formation of natural forests in most of the Mediterraneanfloristic region, but also in sun-exposed areas of theEurosiberian region (Fig. 1). Q. ilex forests can be regardedas one of the rare cases of woodlands that have undergonevery low or no silvicultural management in Mediterraneanareas. Nevertheless, these areas co-occur in Spain with holmoak dehesawoodlands (i.e., Bhuman-made^ ecosystems char-acterized by a savannah-like structure) that play an essentialrole in economy and ecology. The economic importance de-rives from pastoral resources (e.g., grass, acorns) and productswith commercial value: fuelwood, charcoal, tannins, and as-sociated hunting species (Parsons 1962). The ecologicalimportance also lies in the diversity and singularity of thespecies and vegetation communities that they harbor (Díazet al. 2003). The holm oak is indeed the dominant tree of theoptimum (climax) lowland forest in an altitudinal strip be-tween 300 and 800 m (Sainz et al. 2010).

Global climate change, habitat fragmentation, environmen-tal degradation, and direct management of plant resources are

factors that can have profound evolutionary implications forplants. Genetic diversity is essential to evolve in a changingenvironment and to ensure long-term persistence and survivalfor any species (Frankel and Bennett 1970). Theory predictsthat species with higher genetic diversity may undergo a lowerrisk of inbreeding depression, an increased fitness throughheterozygote benefits, and a better evolutionary potential thanspecies with lower genetic diversity (Frankham et al. 2002;Sherwin and Moritz 2000). Indeed, empirical results haveshown that low genetic diversity may be responsible for theextinction of populations and species (e.g., Saccheri et al.1998; Újvária et al. 2002). Hence, studies addressing levelsof genetic diversity can help to reduce the risks of loss ofbiodiversity, by identifying populations in a critical state, solv-ing taxonomic uncertainties, defining management units with-in species, detecting hybridization, defining locations for re-introduction programs, and selecting the most adapted popu-lations to specific environments (Frankham 2005). The impor-tance of the conservation of genetic diversity was alreadyacknowledged by an explicit goal of the 1993 Conventionon Biological Diversity. Current protected areas, such as na-tional parks, nature reserves, and wilderness areas (UNEP-

Fig. 1 Geographical distribution of the 39 ptDNA haplotypes detected inthe survey inQuercus ilex from Spanish populations. Pie charts representthe proportion of individuals observed for each haplotype of eachpopulation (Table 2), with chart size proportional to the number of

individuals analyzed. Population codes are identified in Table 1. Protectedareas with codes in bold, refugia in italic, or both. Inset in the right bottomcorner is the distribution of Q. ilex in Spain based on Maldonado et al.(2001)

124 Page 2 of 18 Tree Genetics & Genomes (2015) 11: 124

WCMC 2008), could help to conserve the genetic diversity ofspecies by capturing the maximum genetic diversity of speciesgene pools (Maxted et al. 2008). Traditionally, Spanish poli-cies and management regimes for natural areas have beendesigned for the conservation of flagship species. The protec-tion of natural areas could help, secondarily, to conserve manyother species with nonsingular biological characteristics butthat contribute significantly to regional biodiversity and eco-system functioning. This added value (i.e., beyond flagshipspecies) could be defined in terms of the contribution ofprotected areas to the preservation of the genetic diversity ofnonthreatened, widely distributed species that are neverthelesseconomically and/or socially important. Q. ilex is a widelydistributed and economically important keystone species inMediterranean ecosystems, with some populations located inSpanish protected areas (7 national parks and 338 natureparks), and therefore offers the opportunity to evaluate theimportance of protected areas as reservoirs of genetic diversityusing nonflagship species.

As regards conservation issues, areas with high geneticdiversity should constitute high priority conservation areas.Glacial refugia are subject to intense scientific research(Shafer et al. 2010 and references therein; Weiss and Ferrand2007 and references therein) because long-term isolated pop-ulations offer unique genotypes (Hewitt 1996; but see Petitet al. 2003). A general assumption inherent to most studiesis that glacial refugia harbor higher levels of genetic diversitythan do areas that have been recently colonized after the retreatof glaciers and cold areas because refugia generally representpart of the original gene pool (Comes and Kadereit 1998;Taberlet et al. 1998). In the last few years, several studies havebeen published on this subject, where fossil pollen, macro-fossil, and organellar markers have been used to clarify ourunderstanding of postglacial recolonization. Under this ap-proach, glacial refugia in the Iberian Peninsula have beenproposed for three evergreen oak species (López de Herediaet al. 2007a). The complex orography and geographicmosaic of habitats in the Iberian Peninsula have favoredthe occurrence of multiple refugia. Fifty-two putativerefugia of plants were identified in the Mediterraneanfloristic region (Médail and Diadema 2009), including nineSpanish refugia. In this paper, we test the explicit hypothe-sis of nine glacial refugia in Spain using nuclear and plastidmolecular markers.

International efforts to improve the management of treegenetic diversity were initiated more than 40 years ago(Palmberg-Lerche 2007) focusing on forest genetic resources,i.e., genetic variation in trees valuable for present or futurehuman use (FAO 1989). In Europe, The European ForestGenetic Resources Programme (EUFORGEN) has been facil-itating international collaboration to promote the effectiveconservation and sustainable use of forest genetic resourcesover 20 years (www.euforgen.org, Koskela et al. 2014;

Lefèvre et al. 2013). The EUFORGEN Mediterranean oaksnetwork (Bozzano and Turok 2003) and the Spanish govern-ment, through its Strategy for Conservation and SustainableUse of Forest Genetic Resources (Jimenez et al. 2009), pro-mote gene conservation of Q. ilex, Quercus pubescens, andQuercus suber. In situ conservation projects (creatingprotected areas, seed stands, or gene conservation forests)and technical guidelines for gene conservation have been de-veloped in order to preserve oak species (Bozzano and Turok2003). The network emphasized, however, that little knowl-edge about genetic resources of Mediterranean oaks was amain constraint for their conservation and enhancement.Techniques in molecular genetics have proven to be powerfultools to assess the level and distribution of genetic diversityacross populations. In addition, plastid DNA (ptDNA), a mol-ecule maternally inherited in oaks (Dumolin et al. 1995), andnuclear markers have been shown to be a powerful tool forestimating intraspecific levels of genetic diversity. Isozymemarkers (Michaud et al. 1992) and ptDNA RFLPs (Lumaretet al. 2002) were used to asses genetic variation anddifferentiation among populations of holm oak across theMediterranean region. Genetic diversity has particularly beenassessed in Q. ilex from the Italian Prealps (Vernesi et al.2012) and from the Iberian Peninsula and Balearic Islands(López de Heredia et al. 2007b) using amplified fragmentlength polymorphisms (AFLPs), nuclear rDNA ITS, andptDNA RFLPs. Protected areas and glacial refugia are sup-posed to preserve a substantial fraction of the genetic diversityof the species. Considering all this, we conducted a geneticsurvey of Q. ilex populations to estimate the level and distri-bution of neutral genetic diversity of populations throughoutSpain. Our ultimate objective is to determine whetherprotected natural areas and/or putative glacial refugia are es-sential to maintain the neutral genetic diversity of Q. ilex inSpain. To this end, we compared the genetic diversity betweenprotected/nonprotected and refugial/nonrefugial areas.

Materials and methods

Study system

Protected areas cover a relatively large part of Europe (21% ofthe European Union territory) and are designed to conserve abroad spectrum of species and ecosystems. Europe has a com-paratively high percentage of protected areas since setting upthe largest network of protected areas (Natura 2000 network):in particular, 30 % of Spain’s national land territory is coveredby Natura 2000 network sites. In terms of absolute land area,Spain significantly provides the largest protected area of thisnetwork (c. 139 million ha) in Europe. On the other hand, theSpanish national network of protected areas is composed of1740 locations (6.9 million ha, 12.85 % of the Spanish

Tree Genetics & Genomes (2015) 11: 124 Page 3 of 18 124

http://www.euforgen.org/

territory) (EUROPARC-España 2012). The overlap betweenSpanish and Natura 2000 network of protected areas is ap-proximately 42 %. In particular, the Spanish national networkincludes 15 national parks (0.75 % of the territory; 381,261 ha) and 1725 nature parks (i.e., protected areas with alower protection status such as natural parks, regional parks,natural reserves, natural monuments, and natural protectedlandscapes). Q. ilex occupies c. 32,000 ha in seven nationalparks (Cabañeros, Monfragüe, Ordesa yMonte Perdido, Picosde Europa, Sierra de Guadarrama, Sierra Nevada, and Tablasde Daimiel) and c. 751,780 ha in 338 nature parks (Table S1).Sierra Norte de Sevilla and Sierra de Aracena y Picos deAroche Nature Parks are the Spanish protected areas withthe largest area occupied by the holm oak (Table S1,Fig. S1A). Within Spanish National Parks, Cabañeros is theone that protects the higher number of hectares of Q. ilexwoodlands (12,516 ha).

Sampling and DNA extraction

Leaf samples were collected from 68 populations of Q. ilexdistributed across continental Spain and the Balearic Islands(Fig. 1), which included protected/nonprotected (27/41 popu-lations) and refugial/nonrefugial (22/46 populations) areas fora total of 660 individuals (Table 1). Populations were classi-fied as protected/nonprotected based on EUROPARC-España(2012) and as refugial/nonrefugial based on Médail andDiadema (2009). Between 2 and 20 individuals per population(mean for AFLPs=9.29; mean for ptDNA=9.70) were sam-pled. Total genomic DNA was extracted from dried leavesusing the DNeasy Plant Mini Kit (Qiagen, CA) according tothe manufacturer’s instructions. The number of individualsused for ptDNA SNPs, ptDNA microsatellites, and AFLPsfrom each population is found in Table 2.

ptDNA variation and data analysis

To detect variation within the Q. ilex plastid genome, plastidmononucleotide repeat regions were genotyped in 660 indi-viduals using previously developed primers in Weising andGardner (1999) and Sebastiani et al. (2004). PCR was under-taken in a total volume of 5 μl consisting of 1x PCR buffer,0.5 mM of each dNTP, 50 mMMgCl2, 10 mM of each primer(the shorter or forward primer was 6-FAM labeled), and 0.1 UEcoTaq Taq polymerase (Bioline). PCR products were diluted1:100 before electrophoresis on an Applied Biosystems 3730Genetic Analyzer, and allele sizes were scored usingGeneMapper 4.1 software (Applied Biosystems Inc.). SeeTable S2 for primers’ sequences.

In addition, due to the paucity of intraspecific variationdocumented in public databases for Q. ilex, we sequenced11 plastid DNA regions (atpB-rbcL, atpH-atpI, psbC-trnS,trnC-trnD, trnD-trnT, trnH-psbA, trnK-matK, trnS-trnfM,

trnS-trnG, trnS-trnT, trnT-trnL) for a subset of eight individ-uals from different populations in order to detect variable sites.This resulted in the detection of three regions which weresuitable for primer design (atpB-rbcL, psbC-trnS, and trnH-psbA) and genotyping using a modified PCRAmplification ofMultiple Specific Alleles (PAMSA) protocol (Gaudet et al.2007) (see Table S2 for primers’ sequences). All samples(660 individuals) were genotyped via PCR in a total volumeof 10 μl consisting of 1x PCR buffer, 0.5 mM of each dNTP,50 mMMgCl2, 10 mM of each of the three primers, and 0.1 UEcoTaq Taq polymerase (Bioline). Primer-induced ampliconsize variation corresponding to specific alleles was detectedvia gel electrophoresis using 2.5 % Metaphor agarose gelsstained with SybrSafe (Invitrogen).

Haplotype diversity (h) was calculated using the programDnaSP 5.10 (Librado and Rozas 2009). A haplotype mediannetwork was constructed in the program Network v. 4.6(Bandelt et al. 1999). The contribution of haplotype diversityof each population to the total diversity (CT value) was calcu-lated using the program CONTRIB (Petit et al. 1998).

AFLP fingerprinting

A modification of the AFLP methods described by Vos et al.(1995) andWolf et al. (2004) was used to reveal global geneticvariability betweenQ. ilex samples. For each individual, 55 ngof DNAwas digested and ligated for 2 h at 37 °C using 5 U ofEcoRI and 1 U of MseI (New England Biolabs), 0.45 μMEcoRI adaptor, 4.5 μM MseI adaptor (Table S3 for primers’sequences), and 1 U of T4 DNA ligase (Sigma) in 11 μl totalvolume of 1× T4 DNA ligase buffer (Sigma), 1 μl of 0.5 MNaCl, supplemented with 0.5 μl at 1 mg/ml of bovine serumalbumin (BSA). Enzymes were then inactivated by heating to65 °C for 5 min. Adaptors for each enzyme used were pre-pared by mixing the same amount of the two strands of eachadaptor to a concentration of 5 μM for EcoRI and 50 μM forMseI. Mixes were then denatured at 95 °C for 5 min in athermocycler and finally allowed to slowly cool to room tem-perature in a Styrofoam box for complete annealing. Fivemicroliters of restriction/ligation products were size fraction-ated by electrophoresis through a 2.5 % w/v agarose gel toconfirm complete digestion. Restriction/ligation productswere then diluted 1:10 in Tris low EDTA (EDTA=0.1 M)and stored at −20 °C until used.

Restriction and adaptor ligation were followed by two suc-cessive rounds of PCR amplification. For preselective ampli-fication, 3 μl of the diluted restriction/ligation products de-scribed above were incubated in 12.5 μl volumes containing1× Biomix (Bioline, London, UK) with 0.25 μl ofPreampEcoRI primer and 0.25 μl PreampHpaII/MspI (bothprimers at 10 μM) (Table S3 for primers’ sequences) supple-mented with 0.1 μl at 1 mg/ml of BSA. PCR conditions were2 min at 72 °C followed by 30 cycles of 94 °C for 30 s, 56 °C


Tab

le1

Accession

dataforthe68

populatio

nsof

Quercus

ilex(660

individuals)

Populatio

ncode

Locality

Coordinates

Protected

areas

Putativerefugialarea

a

Current

figure

ofprotectio

nArea/area

occupied

byQ.ilex(ha)

Dateof

designation

AAlicante,F

ontR

oja

38.6711,

−0.4851

Font

RojaNaturePark

2298/1632

1987

–

Al1

Alm

ería,R

oadObla-Ohanes,

MonteNegro

37.0843,−2

.7341

Sierra

NevadaNationalP

ark

85,883/5999

1999

Sierra

Nevada/Gata

Al2

Alm

ería,S

ierrade

Gador,F

élix

36.8939,−2

.6197

Sierra

Nevada/Gata

Al3

Alm

ería,C

abode

Gata

36.8506,−2

.1000

Cabode

Gata-NíjarNaturePark

49,512/<0.01

b1987

Sierra

Nevada/Gata

Al4

Alm

ería,S

ierrade

Alham

illa

36.9088,−2

.4042

–Sierra

Nevada/Gata

Al5

Alm

ería,L

aUmbría

37.6667,−2

.1333

Sierra

María-Los

Velez

NaturePark

22,562/6063

1987

–

As1

Asturias,So

miedo

43.0919,−6

.2562

Somiedo

NaturePark

29,0215/1045

1988

–

As2

Asturias,Cueva

delP

indal

43.3977,−4

.5350

––

As3

Asturias,Picosde

Europa

43.2970,−4

.8234

Picosde

EuropaNationalP

ark

63,858/157

1995

c–

Av

Ávila,N

avam

ediana

40.3201,−5

.4182

Sierra

deGredosNaturePark

86,440/<0.01

1996

Sistem

aCentral

Ba1

Badajoz,R

otade

laSierra,V

illar

delR

ey39.0849,−6

.8891

––

Ba2

Badajoz,H

ornachos

38.4786,−5

.8942

––

Ca

Cádiz,G

razalema

36.7458,−5

.3551

Sierra

deGrazalemaNaturePark

53,411/18,781

1985

Cadiz/Algeciras

region

Cc1

Cáceres,M

onfragüe

39.8219,−6

.0478

Monfragüe

NationalP

ark

18,396/3679

2007

d–

Cc2

Cáceres,V

egaviana

40.0148,−6

.7266

––

Cc3

Cáceres,H

erreruela

39.4592,−6

.9027

––

Cr1

CiudadReal,ElC

alminar

39.1336,−3

.7418

Tablas

deDaimielN

ationalP

ark

1890/187

1973

–

Co1

Córdoba,O

bejo,R

íoGuadalbarbo

38.0971,−4

.8371

––

Co2

Córdoba,betweenCarcabuey

andRota

37.4222,−4

.3004

SierrasSubbéticas

NaturePark

32,055/3594

1988

–

Co3

Córdoba,V

entadelC

harco

38.2539,−4

.31899

Sierra

deCardeña

yMontoro

NaturePark

38,449/21,845

1989

–

Co4

Córdoba,E

lChinche,V

illanueva

deCórdoba

38.3508,−4

.5647

––

Cr2

CiudadReal,Brazatortas

38.6914,−4

.2542

––

Cr3

CiudadReal,Pu

eblade

Don

Rodrigo

39.1177,−4

.5694

––

Cr4

CiudadReal,Navas

deEstena

39.4953,−4

.5209

––

Cr5

CiudadReal,MonteCabañeros

39.3694,−4

.5188

Cabañeros

NationalP

ark

40,828/12,516

1995

e–

Cr6

CiudadReal,LaRaña

39.3318,−4

.3438

Cabañeros

NationalP

ark

40,828/12,516

1995

e–

Gi

Girona,CaboNorfeu

42.2409,3.2618

Cabode

Creus

NaturePark

13,922/22f

1998

–

Gr1

Granada,L

aBordaila

36.9384,−3

.4868

Sierra

NevadaNationalP

ark

85,883/5999

1999

Sierra

Nevada/Gata

Gr2

Granada,S

ierraNevada

37.1393,−3

.5127

Sierra

NevadaNaturePark

86,355/11,359

1989

Sierra

Nevada/Gata

Gr3

Granada,V

entasde

Zafarraya

36.9603,−4

.0285

––

Gr4

Granada,P

ilasde

Algaida

36.9590,−4

.0873

––


Tab

le1

(contin

ued)

Populatio

ncode

Locality

Coordinates

Protected

areas


a

Current

figure

ofprotectio

nArea/area

occupied

byQ.ilex(ha)

Dateof

designation

H1

Huelva,Cabezas

Rubias

37.7303,−7

.0700

––

H2

Huelva,road

toPuertoMoral

37.8624,−6

.4839

Sierra

deAracena

ypicosde

ArocheNaturePark

186,823/97,082

1989

–

H3

Huelva,road

toEncinasola

38.0004,−6

.7540

Sierra

deAracena

ypicosde

ArocheNaturePark

186,823/97,082

1989

–

Hu1

Huesca,Agüero

42.3675,−0

.8123

–S.P

yrenees

Hu2

Huesca,Ordesa

42.6021,−0

.0059

–S.P

yrenees

Hu3

Huesca,Añisclo

42.5167,0.1043

OrdesayMontePerdido

NationalP

ark

15,692/133

1918

S.Py

renees

Ib1

BalearicIslands,Ibiza,SantaEulalia

delR

ío38.8958,1.3349

–BalearicIslands

Ib2

BalearicIslands,Mallorca,Fo

rmentor

39.9147,3.0235

–BalearicIslands

Ib3

BalearicIslands,Menorca,A

lbufera

Des

Grau

39.9333,4.2333

AlbuferaDes

GrauNaturePark

5227/43

1995

BalearicIslands

Ib4

BalearicIslands,Menorca,roadto

Mahon

from

Des

Graus

39.9000,4.3167

–BalearicIslands

J1Jaén,C

uevasBermejas

37.9650,−2

.8515

Sierrasde

Cazorla,S

egurayLas

Villas

NaturePark

210,066/3671

1986

Sierra

Cazorla/Segura

J2Jaén,S

ierraSur,F

uensantade

Martos

37.6511,−3

.9217

––

J3Jaén,S

ierrade

Andújar

38.1309,−3

.9835

Sierra

deAndújar

NaturePark

74,774/7964

1989

–

J4Jaén,T

orreperogil

38.0312,−3

.3408

––

Le

León,VillafrancadelB

ierzo

42.6136,−6

.8150

––

Lu

Lugo,Cruzal

42.8466,−7

.1367

––

M1

Madrid,Navalagam

ella

40.4675,−4

.1323

–SistemaCentral

M2

Madrid,TresCantos

40.6033,−3

.6716

CuencaaltadelM

anzanares

NaturePark

42,583/12,025

1985

Sistem

aCentral

Ma1

Málaga,Sierra

delasNieves

36.7778,−5

.0670

Sierra

delasNievesNaturePark

20,163/896

1989

Serraníade

Ronda

Ma2

Málaga,Mijas,Alhaurínde

laTo

rre

36.6495,−4

.5955

–Serraníade

Ronda

Ma3

Málaga,Casabermeja

36.8939,−4

.4168

–Serraníade

Ronda

Sa1

Salamanca,V

aldunciel

41.1151,−5

.6761

––

Sa2

Salamanca,V

alero

40.5052,−5

.9501

–SistemaCentral

Sa3

Salamanca,P

ozos

delP

uerto

40.5052,−5

.9501

––

Se1

Seville,S

ierraGuillena

37.5914,−6

.0325

––

Se2

Seville,P

inares

deAznalcazar

37.2261,−6

.1905

––

Se3

Seville,betweenConstantin

aand

ElP

edroso

37.8517,−5

.6822

Sierra

Nortede

Sevilla

Nature

Park

177,483/103,370

1989

–

Se4

Seville,M

orón

delaFrontera

37.2278,

−5.4272

––


Tab

le1

(contin

ued)

Populatio

ncode

Locality

Coordinates

Protected

areas


a

Current

figure

ofprotectio

nArea/area

occupied

byQ.ilex(ha)

Dateof

designation

SgMontejo

delaVega

41.5462,−3

.6512

Hoces

delR

íoRiaza

NaturePark

5185/12,516

2005

g–

Te2

Teruel,E

lArdal

40.6170,−1

.4644

––

Te3

Teruel,L

aCuerda

40.5500,−1

.4167

––

Te4

Teruel,S

anGinés

40.6305,−1

.4675

––

To1

Toledo,L

asVentasconPeña

Aguilera

39.5946,−4

.2116

––

To2

Toledo,roadto

Velada

39.9156,−4

.9817

––

VValencia,Sierra

delaMurta

39.0254,−0

.3078

–Valenciaregion

ZZam

ora,Escobar

deTábara

41.7840,−5

.9881

––

aPu

tativ

eglacialrefugiadefinedin

Médailand

Diadema(2009)

bMarinehectares=12,012

cDesignatedas

Montaña

deCovadonga

NationalP

arkin

1918

dDesignatedas

nature

park

in1979

eDesignatedas

nature

park

in1988

fMarinehectares=3073

gDesignatedas

Birds

ofPrey

Refugeof

Montejo

delaVegain

1974


Table 2 Genetic diversity within 68 populations ofQuercus ilex based on AFLP (215 markers) and ptDNA (three SNP loci and four microsatellites).Population codes are identified in Table 1

Population code AFLPs ptDNA

N NPL Aa Apa PLP 1 %b Hj DW N Haplotypes

Protected

A 10 83 1.12 0.02 32.6 0.118 2.642 10 H1,H2

Al1 10 93 1.13 0.03 38.1 0.126 4.961 10 H5,H10,H11

Al3 3 50 1.09 0.02 13.0 0.111 0.921 3 H3,H4

Al5 7 82 1.13 0.02 32.6 0.128 2.698 9 H2,H5

As1 10 99 1.13 0.03 41.4 0.125 5.010 10 H3,H6

As3 10 77 1.10 0.02 29.8 0.100 4.399 7 H12

Av 10 89 1.11 0.03 36.3 0.109 4.898 10 H12,H14,H15,H16

Ca 10 85 1.12 0.02 34.4 0.116 3.123 10 H2,H5,H20,H21

Cc1 10 85 1.13 0.03 35.8 0.123 3.359 10 H12

Co2 9 82 1.12 0.02 33.5 0.120 2.420 10 H5,H10

Co3 8 93 1.14 0.03 39.1 0.135 5.517 10 H2,H5,H11

Cr1 12 58 1.12 0.02 37.2 0.112 3.866 12 H2,H5

Cr5 9 89 1.13 0.03 35.0 0.123 3.916 10 H5,H22

Cr6 10 80 1.11 0.02 33.0 0.110 2.677 10 H5,H22,H23

Gi 8 70 1.10 0.02 26.5 0.104 2.204 10 H2,H5,H25

Gr1 10 91 1.13 0.02 37.2 0.119 5.236 10 H9,H27

Gr2 10 96 1.14 0.04 41.9 0.131 5.370 10 H5,H9

H2 8 79 1.12 0.02 31.2 0.120 2.566 10 H2

H3 10 85 1.12 0.02 35.8 0.117 2.614 10 H29

Hu3 10 100 1.14 0.02 41.0 0.127 3.868 10 H30,H31

Ib3 4 53 1.10 0.02 18.6 0.107 1.057 6 H6

J1 10 95 1.13 0.03 39.5 0.127 4.424 10 H33

J3 8 90 1.13 0.03 35.8 0.134 3.670 10 H2,H5,H11

M2 8 80 1.12 0.02 30.7 0.111 2.515 9 H2,H5

Ma1 10 103 1.14 0.03 42.0 0.132 5.613 10 H2,H5,H35

Se3 10 78 1.10 0.01 31.2 0.107 2.174 10 H5

Sg 10 98 1.13 0.03 39.5 0.123 5.099 10 H2

Mean (SD) – 83.81 (13.50) 1.21 (0.01)c 0.02 (0.006)c 34.2 (6.60)c 0.119 (0.01)c 3.586 (1.358)c – –

Total 244 65 1.38 0.13 – – – 256 H1-H6, H9-H12,H14-H16, H20-H23,H25, H27, H29, H30,H31, H33, H35

Non-protected

Al2 9 86 1.12 0.02 34.4 0.120 2.715 10 H9

Al4 10 78 1.12 0.01 30.7 0.115 2.293 10 H6,H7,H8

As2 9 77 1.10 0.03 30.7 0.105 3.330 10 H12,H13

Ba1 10 84 1.12 0.03 34.0 0.120 5.537 10 H17

Ba2 10 94 1.12 0.03 38.1 0.119 5.590 10 H2

Cc2 9 78 1.11 0.02 31.2 0.114 2.388 10 H19

Cc3 10 82 1.12 0.03 32.6 0.119 3.718 10 H1,H17,H18

Cr2 10 81 1.12 0.02 34.0 0.114 2.914 10 H2

Co1 10 91 1.13 0.03 38.1 0.126 3.982 10 H5,H11,H24

Co4 10 90 1.13 0.02 36.7 0.124 4.052 10 H2,H5,H11

Cr3 10 97 1.12 0.03 39.1 0.116 6.525 10 H5,H10

Cr4 9 89 1.12 0.02 35.8 0.117 2.797 9 H5

Gr3 9 86 1.13 0.02 34.0 0.122 3.796 10 H2,H26

Gr4 11 60 1.11 0.02 35.8 0.109 5.056 11 H2,H26


Table 2 (continued)

Population code AFLPs ptDNA

N NPL Aa Apa PLP 1 %b Hj DW N Haplotypes

H1 9 95 1.14 0.03 39.1 0.129 6.017 10 H5,H28

Hu1 19 75 1.12 0.02 46.0 0.109 6.261 20 H6

Hu2 10 90 1.12 0.03 36.7 0.119 4.886 10 H30,H31

Ib1 14 71 1.14 0.03 44.7 0.122 7.823 18 H2,H6,H32

Ib2 10 82 1.11 0.03 33.0 0.107 6.984 10 H1,H2,H7,H17

Ib4 3 56 1.11 0.04 16.7 0.124 2.512 4 H6,H26

J2 10 102 1.15 0.04 44.2 0.135 5.519 10 H2,H5,H11,H34

J4 10 73 1.08 0.02 28.4 0.101 2.646 9 H5

Le 10 87 1.12 0.02 34.4 0.119 3.742 10 H19

Lu 9 83 1.12 0.02 33.5 0.116 2.427 10 H19

M1 10 101 1.13 0.03 40.0 0.123 6.501 10 H2

Ma2 10 81 1.11 0.02 32.6 0.112 3.148 10 H6

Ma3 10 86 1.13 0.02 36.3 0.123 2.742 10 H1,H2,H29

Sa1 2 42 1.09 0.01 8.8 0.103 0.240 2 H6

Sa2 9 92 1.12 0.02 36.7 0.122 3.196 10 H1

Sa3 10 88 1.12 0.01 34.4 0.117 5.291 10 H1,H3

Se1 10 87 1.12 0.02 34.9 0.114 3.532 10 H29,H36

Se2 9 86 1.12 0.02 35.3 0.116 2.801 10 H5,H15

Se4 10 85 1.12 0.02 34.0 0.119 3.512 10 H37,H38

Te1 10 89 1.11 0.03 35.8 0.106 5.280 10 H6,H39

Te2 7 78 1.13 0.03 30.7 0.122 2.229 7 H6

Te3 7 74 1.12 0.02 29.8 0.111 1.548 7 H6,H39

Te4 6 65 1.10 0.01 23.7 0.105 1.790 7 H6,H39

To1 10 78 1.11 0.02 31.6 0.109 3.780 10 H5,H23

To2 10 95 1.13 0.02 38.1 0.121 4.412 10 H5,H12,H18

V 8 77 1.12 0.02 28.8 0.114 3.174 10 H1,H2

Z 10 90 1.13 0.02 36.3 0.122 3.212 10 H12

Mean (SD) – 82.46 (11.80) 1.12 (0.01)c 0.02 (0.007)c 33.86 (6.54)c 0.116 (0.007)c 3.899 (1.649)c – –

Total 388 66 1.38 0.13 – – – 404 H1-H3, H5-H13, H15,H17-H19, H23, H24,H26, H28-H32, H34,H36-H39

Refugia

Mean (SD) – 82.91 (14.91) 1.12 (0.01)c 0.02 (0.007)c 34.23 (8.66)c 0.118 (0.008)c 4.158 (1.864)c – –

Total 207 67 1.39 0.14 – – – 220 H1-H12, H14-H17,H20, H21, H26, H27,H30-H33, H35

Non-refugia

Mean (SD) – 83.04 (11.23) 1.11 (0.01)c 0.02 (0.006)c 33.90 (5.35)c 0.117 (0.008)c 3.592 (1.383)c – –

Total 425 67 1.37 0.12 – – – 440 H1-H3, H5, H6,H10-H13, H15,H17-H19, H22-H26,H28, H29, H34,H36-H39

In italics are populations located in putative glacial refugia according to Médail and Diadema (2009)

N number of individuals, NPL number of polymorphic loci, A allelic richness, Ap private allelic richness, PLP percentage of polymorphic loci at 1 %level, Hj Nei’s gene diversity (= expected heterozygosity), DW rarity indexaA and Ap were calculated using HP-Rare (Kalinowski 2005) with rarefaction to two samples per population and five populations per levelb PLP calculated using AFLPDIV (Coart et al. 2005; Petit et al. 1998) with rarefaction to 2cMeans not significantly different from each other (Student’s t for Hj and DW; nonparametric k-sample median test for A, Ap, and PLP, p<0.05)


for 30 s, and 72 °C for 2 min with a final extension step of10 min at 72 °C. Five microliters of preselective amplificationproducts were size fractionated by electrophoresis through a2.5 % w/v agarose gel to confirm amplification. PCR productswere then diluted 1:10 in Tris low EDTA (EDTA=0.1 M) andstored at −20 °C until used. Selective PCR reactions were per-formed using 3 μl of the diluted preselective PCR reactionproduct and the same reagents as the preselective reactions butusing 6-FAM or VIC-labeled selective EcoRI primers (Table S3for primers’ sequences). Cycling conditions for selective PCRwere as follows: 94 °C for 2 min, 13 cycles of 94 °C for 30 s,65 °C (decreasing by 0.7 °C each cycle) for 30 s, and 72 °C for2 min, followed by 24 cycles of 94 °C for 30 s, 56 °C for 30 s,and 72 °C for 2 min, ending with 72 °C for 10 min. For eachindividual, 0.5 μl of 6-FAM-labeled and 0.5 μl of VIC-labeledselective PCR products were combinedwith 0.5μl of GeneScan500 LIZ (Applied Biosystems, Foster City, CA, USA) and13.5 μl of formamide. Samples were heat denatured at 95 °Cfor 3–5 min and snap cooled on ice for 2 min. Samples werefractionated on an ABI PRISM 3100 at 3 kV for 22 s and at15 kV for 45 min. Initially, 48 selective primer combinationswere analyzed in a subset of five samples comprising fourpopulations selected to cover most of the species distribution.One replicate was included to test for reproducibility.

AFLP profiles were analyzed using GeneMapper 4.1 soft-ware (Applied Biosystems, Foster City, CA). Two primercombinations (EcoRI-AGG/MseI-CGA and EcoRI-ACT/MseI-CTA) were chosen based on the number of polymorphicmarkers and the level of reproducibility. Markers <100 bp inlength were removed from the data as these showed someevidence of size homoplasy as detected using the method ofVekemans et al. (2002) as implemented in the software AFLP-SURV 1.0. All ambiguous markers and singletons were ex-cluded from the dataset prior to analyses.

AFLP data analysis

Genetic diversity and population genetic structure analyseswere performed using AFLP-SURV ver. 1.0 (Vekemans et al.2002). Allelic frequencies at the AFLP loci were computedfrom the observed frequencies of fragments using theBayesian approach with nonuniform prior distribution of allelefrequencies proposed by Zhivotovsky (1999) for diploid spe-cies, assuming Hardy-Weinberg equilibrium. Based on thesecomputations of allelic frequencies, genetic diversity andpopulation genetic structure were estimated using the approachof Lynch and Milligan (1994). The number of polymorphicloci (NPL) and Nei’s gene diversity (= expected heterozygos-ity) (Hj) were calculated for each population. The percentage ofpolymorphic loci (PLP) was computed by the rarefaction ap-proach to account for unequal sample sizes (2–19, Table 2)using the program AFLPdiv (R. Petit, INRA-Bordeaux,website http://www.pierroton.inra.fr/genetics/labo/Software).

We performed standardization to the smallest sample size(Coart et al. 2005; Petit et al. 1998). We further used theprogram HP-Rare 1.1 (Kalinowski 2005), which enables usersto conduct hierarchical rarefaction. We estimated the allelicrichness (A) and private allelic richness (Ap) based on fivepopulations within each group and two individuals per popu-lation. A and Ap were estimated in protected/nonprotected andrefugial/nonrefugial sets of populations. The Rarity 1 index(equivalent to the frequency down-weighed marker value(DW) according to Schönswetter and Tribsch (2005)) was cal-culated using the R script AFLPdat (Ehrich 2006).

Genetic structure was examined at different levels: (1) allpopulations together, (2) between and within protected/nonprotected populations, and (3) between and withinrefugial/nonrefugial populations. To this end, we estimatedtotal gene diversity (Ht), mean gene diversity within popula-tions (Hw), average gene diversity between populations (Hb),and Wright’s fixation index (FST). The significance of geneticdifferentiation between populations in protected/nonprotectedareas, on one hand, and refugial/nonrefugial areas, on the oth-er, was tested by comparing the observed FST with the distri-bution of FST under the null hypothesis of no genetic structureobtained by means of 10,000 random permutations of individ-uals between populations. Population Sa1 (two individuals)was excluded for this analysis. The significance of the ob-served differences between the genetic diversity indices ofpopulations from protected/nonprotected and refugial/nonrefugial areas was tested by Student’s t test except for A,Ap, and PLP that could not be transformed to normality; there-fore, the nonparametric k-sample median test was performed.Both significance tests were performed with IBM SPSSStatistics v. 21. Additionally, a hierarchical analysis of molec-ular variance (AMOVA, Excoffier et al. 1992) with popula-tions nested within levels of area protection was performedusing the software GenALEX 6.501 (Peakall and Smouse2006) to examine the distribution of total genetic variationand differential connectivity among populations, levels of areaprotection, and populations within levels of area protection.Significance of variance components was based on 9999 per-mutations. The same procedure was performed nesting popu-lations according to their location in putative glacial refugia.

In addition, a distance matrix based on Nei–Li distances (Neiand Li 1979) was used to estimate a neighbor-joining pheno-gram (NJ, Saitou and Nei 1987) in PAUP* version 4.0b10(Swofford 2002). Bootstrap support was estimated based on10,000 replicates using the NJ algorithm. Because of the lackof outgroup samples, midpoint rooting was performed. Aprincipal coordinate analysis (PCoA) conducted in GenALEX6.501 (Peakall and Smouse 2006) on the genetic Nei–Li dis-tancematrix was performed in order to assess the dimensionalityof data and visualize the dispersion of individual plants.

Our low sample size of some populations (Table 2) couldbias the results. For this reason, we performed all the analyses


http://www.pierroton.inra.fr/genetics/labo/Software

using two datasets: dataset A, that includes the 63 sampled pop-ulations, and dataset B, that includes populations with a samplesize of nine or more individuals (54 populations) (Table 2).

Results

PtDNA haplotype diversity

Three SNP loci and four microsatellite loci (atpF intron, ORF77–82, ndhG-ndhI, and ndhH-rpS15) were variable. Based onthese polymorphisms, 39 ptDNA haplotypes were identified(Table S4) across populations. H2 (20.60 % of 660 individ-uals), H5 (18.50 %), H6 (12.90 %), H12 (7.40 %), and H1(5.40 %) were the most frequent haplotypes (Table S4,Fig. S2) and showed a widespread geographical distribution(Fig. 1). We consider the remaining haplotypes to be geo-graphically restricted and representing rare variants (frequen-cies below 4.2 %). Of the 68 populations, 44 were polymor-phic (with a maximum number of four haplotypes), whereas24 were fixed for particular haplotypes (Table S4, Fig. 1). Thehaplotype median network showed a high number of loopsneeded to connect haplotypes (Fig. S2).

Protected/nonprotected areas A similar number of haplo-types was found in protected (24) and nonprotected (29) pop-ulations (Table 2). Ten haplotypes (H4, H14, H16, H20, H21,H22, H25, H27, H33, and H35) were exclusive to individualsfrom protected populations and occur in single populations(except H22 that occurs in populations Cr6 and Cr7), at lowfrequencies (<1.5 %) (Table S4). Fifteen haplotypes (H7, H8,H13, H17, H18, H19, H24, H26, H28, H32, H34, H36–H39)were exclusive to individuals from nonprotected populationsand occur mostly in single populations (Table 2), at low fre-quencies (<7.6 %) (Table S4). Both protected andnonprotected populations were either monomorphic orshowed up to four different haplotypes (Table 2, Fig. 1). Thecontribution of population haplotypes to the total diversity (CT

value) was positive or negative irrespective of the area status(protected vs. nonprotected) of the populations (Fig. 2).Eleven protected (c. 41 %) and 16 nonprotected (c. 39 %)populations contribute positively to this total diversity value.The positive contributions were driven primarily by theirstrong divergence (Fig. 2). When considering the levels ofhaplotype diversity in a chronological order of protected areadesignation in Spain, a clear stepwise pattern of higher diver-sity is found. The cumulative number of haplotypes inSpanish protected areas (created between 1918 and 1999) isshown in Fig. S2B. In particular, the designation of Cabañerosas Nature Park in 1988 together with Somiedo and SierrasSubbéticas Nature Park and a set of protected areas in 1989,including Sierra de Andújar, Sierra de Aracena y picos deAroche, Sierra de Cardeña y Montoro, Sierra Nevada, Sierra

de las Nieves, and Sierra Norte Nature Parks, resulted in aconsiderable rise of the number of haplotypes in protectedareas. As summarized in Table S4, in protected areas,ptDNA haplotype diversity (h=0.836) was similar than innonprotected areas (h=0.898).

Refugial/nonrefugial areas Fifty-one haplotypes were foundin refugial (26 haplotypes) and nonrefugial (25 haplotypes)populations (Table 2). Fourteen haplotypes (H4, H7–H9,H14, H16, H20, H21, H37, H30–H33, and H35) were exclu-sive to individuals from refugial areas and occur in singlepopulations (except H9 that occurs in three populations andH7, H30, and H31 in two populations), at frequencies lowerthan 12.7% (Table S4). In addition, fourteen haplotypes (H13,H18, H19, H22–H25, H28, H29, H34, H36–H39) were ex-clusive to individuals from nonrefugial areas and occur insingle populations (except H19 and H39 that occur in threepopulations and H18, H22, H23, and H29 in two populations),at frequencies lower than 6.8 % (Table S4). A similar percent-age of populations from refugial (c. 41 %) and nonrefugial (c.36 %) areas showed a high contribution to the total CT valuediversity (Fig. 2). This was due mostly to the own divergencecomponent. Similar haplotype diversity was found in popula-tions from refugial (h=0.876) and nonrefugial (h=0.866)areas (Table S4).

AFLP primer combinations

The pilot study for the choice of primer combinations yieldeda percentage of polymorphic markers between 43.5 and85.1 %, and the reproducibility of the observed markersranged between 62.5 and 100 % (Table S5). Two AFLP se-lective primer combinations resulted in 215 unambiguousfragments when extended to the successfully genotyped sam-ple of 632 individuals (68 populations). All these fragmentswere polymorphic. The primer combination (EcoRI-AGG/MseI-CGA) yielded 66 markers in the range of 103–269 bpand EcoRI-ACT/MseI-CTAyielded 149 markers in the rangeof 100–500 bp. The reproducibility of the observed AFLPfragments was 98.6 % for both primer combinations.

AFLP genetic diversity

The results obtained from both datasets (dataset A, that in-cludes all the 68 sampled populations, and dataset B, thatincludes 54 populations with sample sizes equal or largestthan 9) did not show significant differences. For the sake ofbrevity, we show and discuss only the results from dataset A.See Tables S6–S8 for results from dataset B.

Table 2 summarizes the genetic diversity among 68 popu-lations of Q. ilex. Individual populations had a mean percent-age of polymorphic loci of 34 % ranging from 8.8 % in pop-ulation Sa1 to 46 % in population Hu1 (Table 2). Per-


population Nei’s gene diversity (Hj) (= expected heterozygos-ity), under a model assuming no deviation from Hardy-Weinberg genotypic proportions, ranged from 0.105 (As2) to0.135 (J2), with an average of 0.118±0.008.

Protected/nonprotected areas Populations in protected areashad, on average, only slightly and not significantly higherlevels of gene diversity (Hj=0.119±0.010) than those from

nonprotected areas (Hj=0.116±0.007) (Table 2). The propor-tion of rare AFLPmarkers did not significantly differ betweenpopulations from protected (DW=3.586) and nonprotected(DW=3.899) areas, and it was the highest in the nonprotectedpopulation Ib2 (DW=6.984) (Table 2).

Refugial/nonrefugial areas Per-population Nei’s gene diver-sity (Hj) ranged from 0.100 to 0.132 (average Hw=0.118)

AA

l1A

l3A

l5A

s1A

s3 Av

Ca

Cc1

Co2

Co3

Cr1 Sg

Cr5

Cr6 G

iG

r1G

r2 H2

H3

Hu3

Ib3 J1 J3

M2

Ma1

Se3

Al2

Al4

As2

Ba1

Ba2

Cc2

Cc3

Co1

Co4

Cr2

Cr3

Cr4

Gr3

Gr4 H1

Hu1

Hu2

Ib1

Ib2

Ib4 J2 J4 Le

Lu

M1

Ma2

Ma3

Sa1

Sa2

Sa3

Se1

Se2

Se4

Te1

Te2

Te3

Te4

To

1T

o2 V Z

Con

trib

uti

on

to d

iver

sity

-0.010

-0.008

-0.006

-0.004

-0.002

0.000

0.002

0.004

0.006

0.008

0.010

Differentiation DiversityTotal

Protected populations Non-protected populations

A

Al1

Al2

Al3

Al4 Av

Ca

Gr1

Gr2

Hu1

Hu2

Hu3

Ib1

Ib2

Ib3

Ib4 J1

M1

M2

Ma1

Ma2

Sa2 V A

Al5

As1

As2

As3

Ba1

Ba2

Cc1

Cc2

Cc3

Co1

Co2

Co3

Co4

Cr1

Cr2

Cr3

Cr4

Cr5

Cr6 G

iG

r3G

r4 H1

H2

H3 J2 J3 J4 Le

Lu

Ma3

Sa1

Sa3

Se1

Se2

Se3

Se4 Sg

Te1

Te2

Te3

Te4

To

1T

o2 Z

Con

trib

uti

on

to d

iver

sity

Differentiation DiversityTotal

Refugial populations Non-refugial populations-0.010

-0.008

-0.006

-0.004

-0.002

0.000

0.002

0.004

0.006

0.008

0.010B

Fig. 2 Individual contributions of 68 Quercus ilex populations to global haplotype diversity between protected/nonprotected (a) and refugial/nonrefugial (b) populations. The contribution of each population is subdivided into diversity and differentiation components


within refugial populations and from 0.100 to 0.135 (averageHw=0.117) within populations from nonrefugial areas(Table 3). The intrapopulation genetic diversity indices char-acterized above did not significantly differ between both setsof populations (p>0.05). The proportion of rare AFLPmarkers was higher in refugial (DW=4.158) than innonrefugial (DW=3.592) populations. However, the differ-ence was not statistically significant (Table 2).

AFLP genetic structure

The following show the results of dataset A (see the results ofdataset B in Tables S6–S8). There were significant but lowgenetic differentiation among the studied Q. ilex populations(FST=0.0167, p<0.01; Table 3). The two-dimensional PCoAand the AFLP-based neighbor-joining tree revealedweak pop-ulation differentiation and the absence of a pattern of cluster-ing either by protected/nonprotected (Fig. S3A) or by refugial/nonrefugial (Fig. S3B) areas.

Protected/nonprotected areas Total gene diversity wasslightly higher in the group of protected populations (Ht=0.1210) than in the group of nonprotected populations (Ht=0.1192). There was very low genetic, and not significant, dif-ferentiation between the two levels of protection (FST=0.0004, p=0.21); genetic differentiation among populationswithin protected (FST=0.0152) areas was slightly lower thanwithin nonprotected (FST=0.0178) areas (p<0.0001, Table 3).The hierarchical AMOVA indicated that the level of protec-tion was not a significant source of variability (Table 4) andrevealed that most of the variance was found among individ-uals within populations (95.03 %).

Refugial/nonrefugial areas Total gene diversity was slightlyhigher in the group of populations located in refugial areas(Ht=0.1215) than in the group of nonrefugial populations

(Ht=0.1191). Little genetic differentiation, but significant,was found between refugial/nonrefugial areas (FST=0.0023,p<0.0001) and was higher among populations within refugial(FST =0.0245) than nonrefugial (FST =0.0115) areas(p<0.0001, Table 3). The hierarchical AMOVA showed sig-nificant but low differences between refugial/nonrefugial pop-ulations (0.24 % of the total genetic diversity, p<0.0001;Table 4). Approximately, 5 % of the total genetic diversitywas attributable to population differences within refugial/nonrefugial areas whereas c. 95 % to differences among indi-viduals within a population (Table 4).

Discussion

The molecular diversity detected by ptDNA SNPs, ptDNAmicrosatellites, and AFLPs showed relatively low genetic di-versity levels for the species in Spain and a quite homoge-neous genetic structure across populations of the holm oak.Previous studies of Q. ilex already found a rather homoge-neous ptDNA genetic structure in Spanish populations(López de Heredia et al. 2007b; Michaud et al. 1992). Inaddition, we found levels of AFLP population differentiation(FST=0.0167, p<0.0001) similarly low to those previouslyfound among populations of Q. suber from Portugal (Coelhoet al. 2006), but even lower than those reported within otherQuercus species (California red oaks, Dodd and Kashani2003; Ireland Quercus petraea and Quercus robur, Kelleheret al. 2005; Iran Quercus brantii, Shiran et al. 2011). Long-lived, wind-pollinated woody species also displayed higherlevels of differentiation using allozyme markers (0.07–0.09in average; Hamrick and Godt 1989) than those found forQ. ilex. Outcrossing, anemophilous long-distance pollen dis-persal, acorn transport by animals, and hardiness of the spe-cies in various habitats most probably played a role in homog-enizing allele frequency among populations (Ducousso et al.

Table 3 Genetic differentiation between populations based on 215 AFLP markers found in 632 individuals of Quercus ilex

N/n Ht Hw (SD) Hb (SD) FST Lower 99 % FST Upper 99 % FST

All populations 68/632* 0.1200 0.1178 (0.0010) 0.0022 (0.0005) 0.0167 −0.0136 −0.0058Among levels of protection 2/632** 0.1055 0.1054 (0.0008) 0.0001 (0.0000) 0.0004 −0.0012 0.0016

Protected 28/244 0.1210 0.1192 (0.0017) 0.0018 (0.0007) 0.0152 −0.0155 −0.0031Nonprotected 40/388*** 0.1192 0.1168 (0.0011) 0.0024 (0.0007) 0.0178 −0.0144 −0.0044Among refugial/nonrefugial 2/632** 0.1061 0.1058 (0.0018) 0.0003 (0.0000) 0.0023 −0.0014 0.0018

Refugial 22/207 0.1215 0.1186 (0.0016) 0.0030 (0.0009) 0.0245 −0.0158 0.0008

Nonrefugial 46/425**** 0.1191 0.1174 (0.0012) 0.0016 (0.0006) 0.0115 −0.0144 −0.0062

In parentheses, standard deviations (SD)

N/n number of populations/number of individuals, Ht total gene diversity, Hw average gene diversity within populations, Hb average gene diversitybetween populations; FST Wright’s fixation index, i.e., differentiation between populations; Lower 99 % FST and Upper 99 % FST, critical values at the99 % at the randomization distribution of FST assuming no genetic differentiation between populations, based on 10,000 random permutations

In FST analysis, *67/630, **2/630, ***39/386, ****45/423


1993 ). Fragmentation may lead to long-term genetic isolation(Young et al. 1996). Nevertheless, extensive gene flow acrossthe holm oak range is inferred for Spanish populations despitethat a fragmented landscape is often observed. Long-distancepollen dispersal and the species’ long lifespan (some centu-ries) could have counterbalanced the expected loss of geneticdiversity by human-mediated fragmentation during the lastcenturies. However, the current distribution of genetic diver-sity of Quercus adult trees is likely to represent that existingbefore human-mediated fragmentation (Craft and Ashley2007; Ortego et al. 2010; Petit et al. 2002).

Protected areas and the genetic diversity of Q. ilex

The general trend of low genetic differentiation found at thespecies level could also be observedwhen analyzing protectedareas. In fact, none of the measures of genetic diversity(ptDNA SNPs, ptDNA microsatellites, and AFLPs) differedsignificantly between protected and nonprotected populations(Tables 2, 3, and 4, Fig. 2, Table S4). The ptDNA geneticdiversity (Table 2, Fig. 1) neither showed a clear geographicpattern among protected areas. Some protected areas had pop-ulations that were highly polymorphic, such as those of Sierrade Gredos and Sierra de Grazalema Nature Parks (four haplo-types found in 10 individuals each). The population ofCabañeros National Park (Table 2, Fig. 1) also displayed ahigh number of haplotypes (three from 10 individuals). Thedifferences in ptDNA genetic diversity were consistent withthe significant but very low AFLP genetic differentiationamong populations within protected areas (FST=0.0152,p<0.0001; Table 3).

Our results suggest that preserving the genetic makeup ofthe holm oak from protected areas does not seem to provide anadded value to the levels of genetic diversity found in Spanishnonprotected areas. However, a great deal of the genetic di-versity is conserved by only preserving the 11.23 % of landdesignated as protected areas in Spain, i.e., the four nationaland three nature parks designated before 1987 (Table 1) helpto protect similar levels of genetic diversity than that found in

nonprotected areas (Fig. S1B), despite these seven protectedareas occupy only c. 7.28 % of the Q. ilexwoodlands. Indeed,protected areas where Q. ilex is more abundant and plays anessential ecological role (e.g., Cabañeros, Sierra Nevada,Monfragüe National Parks) hold a substantial fraction of thespecies’ genetic diversity (Fig. S1A). Conservation of theholm oak in protected areas ensures not only preservation ofthe genetic diversity of the species, but also indirect preserva-tion of an essential ecosystem in Iberian areas that supportspopulations of numerous endangered species, such as thewestern imperial eagle (Aquila adalberti), black stork(Ciconia nigra), cinereous vulture (Aegypius monachus),and Iberian lynx (Lynx pardina) (Díaz et al. 2003; Marañón1986; Tellería 2001).

While haplotype diversity is similar between protected andnonprotected areas, the haplotype composition of both groupsof populations is different. Around 40 % ofQ. ilex haplotypesare not conserved in the Spanish national network, of whichnine haplotypes (60 %) are rare. This does not mean that theyhave no conservation value. In fact, the observed differencesin haplotype composition between protected/nonprotectedareas could be due to selection of specific haplotypes upondifferent environmental conditions. Therefore, conservationoutside the current protected areas network would be neces-sary to preserve Q. ilex identity and adaptability. For practicalconservation, complementing neutral genetic data with adap-tive genetic data would enable a better understanding of evo-lutionary history, adaptive uniqueness of populations, andlong-term persistence. Many studies about the adaptive valueof phenotypic variation in Q. ilex could indicate local differ-entiation (e.g., Bussotti et al. 2015 and references herein).There is now mounting evidence that genetic and epigeneticmechanisms allow organisms to respond to fluctuating envi-ronments (Bird 2007; Leinonen et al. 2008; Mousseau et al.1999). For instance, Rico et al. (2014) showed that acclima-tion response to drought stress in Q. ilex correlates to epige-netic modifications (i.e., DNAmethylation). In addition, it hasbeen suggested that genetic variability among Q. ilex popula-tions could explain morphological variability at a large

Table 4 Hierarchical AMOVA based upon AFLP variation surveyed in a total of 68 populations (632 individuals) ofQuercus ilex under two levels ofprotection (nonprotected, protected) and refugial/nonrefugial areas

Source of variation df SS Variance components Total variance (%) p value

Among levels of area protection 1 19.929 <0.001 0.006 0.42

Among populations within levels of area protection 66 1279.977 0.682 4.96 0.0001

Within populations 564 7368.497 13.065 95.03 0.0001

Among refugial/nonrefugial areas 1 29.200 0.034 0.24 0.001

Among populations within refugial/nonrefugial areas 66 1270.706 0.667 4.85 0.0001

Within populations 564 7368.403 13.065 94.91 0.0001

p value estimates are based on 9999 permutations

df degrees of freedom, SS sum of squared deviations


biogeographic scale, where diverse climatic conditions occur(Lumaret et al. 2002; Peguero-Pina et al. 2014).

Do refugial populations exhibit greater genetic diversity?

Médail and Diadema (2009) defined a glacial refugium as Banarea where distinct genetic lineages have persisted through aseries of Tertiary or Quaternary climate fluctuations owing tospecial, buffering environmental characteristics.^Refugia rep-resented climatically stable areas and constituted key areas forthe long-term persistence of species with narrow ecologicalrequirements. Genetic signatures of refugial areas for biodi-versity during glacial stages typically consist of a high allelicand haplotype richness coupled with a high number of privatealleles and haplotypes with respect to more recently colonizedareas (Hewitt 2001; Petit et al. 2003). We found no evidencefrom ptDNA SNPs, ptDNA microsatellites, or AFLPs to sup-port that the 22 Q. ilex populations located in nine proposedglacial refugia in Spain (Médail and Diadema 2009) constitutedivergent genetic groups as expected after isolation (Table 2,Table S4) (but see López deHeredia et al. 2007a). It is true thatsome populations (Hu1, Ib1, Av, Ca) in refugial areas arehighly polymorphic for some genetic values (Table 2,Table S4). However, those populations show similar levelsof genetic diversity to several other populations (J2 or Cr3)that are not included in the proposed glacial refugia (Table 2).Refugia may prove critical in assisting certain species to per-sist through future rapid climate change as they provide adegree of additional resilience (Holling 1996). For such spe-cies, identification and protection of refugia may be essential.However, the diversity and singularity found in the popula-tions herein analyzed from Spanish refugia (see Table 2)showed that their preservation is not essential for conservationplanning when neutral genetic diversity is considered alone.Besides, most populations of Q. ilex herein analyzed fromrefugial areas are already protected (Table 1).

Populations of Q. ilex occur in semiarid to hyperhumidbioclimates across Spain reflecting the ability of this tree towithstand variable thermic, hydric, and substrate conditions(Barbero et al. 1992). With such a wide range of geographicaland ecological distribution, the persistence of the speciesalong its distribution range is not surprising, even during gla-cial periods. Stress tolerance and phenotypic plasticity (i.e.,the ability of a single genotype to produce differentphenotypes in response to changing environments,Bradshaw 1965) may have helped the holm oak to cope withenvironmental variability (Gimeno et al. 2008). It is worthmentioning that holm oak is more cold resistant than othersclerophyllous species which frequently coexist with it, likeQ. suber (Larcher and Mair 1969) for which a restricted dis-tribution to refugia in the eastern and southwestern Iberia dur-ing glacial periods has been proposed (López de Heredia et al.2007a). Glacial retreat in Europe also resulted in a strong

phylogeographic structure within other European Quercusspecies such as the white oaks (Petit et al. 2002).Nevertheless, human-mediated effects can also be proposedfor the observed pattern of relatively homogeneous geneticdiversity of Iberian populations of Q. ilex. Although the holmoakmay have been the subject of initial domestication, there isvery little evidence of anthropogenic translocation ofQuercusspecies (Aldrich and Cavender-Bares 2011). Besides hardi-ness of the holm oak, another possible explanation for the lackof genetic structure in our data is that the waxing and waningof the species distribution during glacial and interglacial pe-riods might have blurred the genetic structure of earlierfounding populations. Admixture of genetic lineages and highdispersal capabilities may have also altered the genetic signa-ture of isolation across Iberia (Jiménez et al. 2004).

Contemporary climate change is similarly influencing spe-cies distribution and population structure, with important con-sequences for patterns of genetic diversity and species’ evo-lutionary potential. Climate change scenarios (IPCC 2007)predict a rise in temperature and changing patterns of precip-itation in the Iberian Peninsula, resulting in increased waterdeficit. Climate changemay lead to changes in the distributionof Q. ilex, expanding its distribution to higher and coolerareas and limiting its success to drier areas. However, thegenetic makeup of the species in Iberia and its inferred histor-ical resilience to environmental changes are the result of wide-spread survival. Species distribution modeling of Q. ilexagrees with our genetic results in which maintenance of wideand continuous potential areas throughout the IberianPeninsula is already occurring (Carnicer et al. 2014) and pre-dicted for the next century (Felicísimo et al. 2011).Nevertheless, under the most severe scenario, a decrease insuitable area is predicted to be more severe in southwesternIberia. The high resistance to desiccation (3% of open stomatatranspiration in Q. ilex vs 30 % in Quercus humilis) and todrought injury (in Q. ilex 15 times delayed than in Q. humilis;Larcher 1960) will also help to maintain the viability of theholm oak without contracting distribution of populations andgenetic diversity.

Conclusions

The holm oak dominates Mediterranean forests and wood-lands and has been exposed to sustainable utilization forthousands of years. Our results agree with previous research(López de Heredia et al. 2007b; Michaud et al. 1992) interms of relatively low genetic diversity of the holm oak inSpain, and are promising in terms of low risk of neutralgenetic diversity loss under climate change. Extensive geneflow appears to have been prevalent to account for the wide-spread levels of genetic diversity across Spain, althoughsome isolation in glacial refugia is revealed by the genetic


makeup of Q. ilex. The same is true in Spanish protectedareas that did not significantly differ to nonprotected areasby any of the measures of levels of neutral genetic diversity.Nevertheless, future studies identifying genes under selec-tion and analyzing their habitats and geographical distribu-tion could reveal an adaptive value in certain populations. Insum, the Spanish national network is favoring the use ofcurrent protected areas for conservation of the holm oak asa practical way of preserving its genetic diversity. However,additional conservation efforts (e.g., sustainable managementof natural stands, clone banks, research plantations, breedingprograms) may be needed for other target species (FAOet al. 2001).

Acknowledgments The authors thank J. Arroyo, J. Bastida, J. Belliure,J. Camarero, M. Díaz, O. Fiz, P. García-Fayos, C. García Verdugo, C.M.Herrera, O. Lozoya, J. Martínez, V. Mirre, J. Pausas, F. Pulido, A.Tribsch and F. Valladares for field assistance; J. Fernández (QuantumGis) for analytical assistance; and H. Sainz and R. Sánchez de Dios forproviding the Quercus ilex distribution map layer. This research wassupported by Fundación Biodiversidad through the project BLos parquesnacionales españoles como reserva genética para la encina (Quercus ilex),el alcornoque (Quercus suber) y el acebuche (Olea europaea)^ to PVandby the Spanish Ministry of Economy and Competitiveness through aJuan de la Cierva fellowship to BG.

Compliance with ethical standards

Conflict of interest The authors declare that they have no competinginterests.

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Data archiving statement

We have submitted the PAMSA SNPs/AFLPs datasets to http://dendrome.ucdavis.edu/TreeGenes database (accession number:TGDR042).


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http://dx.doi.org/10.1007/s11295-015-0950-2

Documents

Protected areas of Spain preserve the neutral genetic diversity of Quercus ilex L. irrespective of glacial refugia