Populationfragmentationleadstoisolationby
distancebutnotgeneticimpoverishmentinthe philopatricLesserKestrel:acomparisonwith
thewidespreadandsympatricEurasianKestrel
MAlcaide1,DSerrano1,JJNegro1,JLTella1,TLaaksonen2,CMu¨ller3,AGal4 and EKorpima¨ki2
1DepartamentosdeEcolog´ıaEvolutivayBiolog´ıadelaConservacio´n,Estacio´n Biolo´gicadeDon˜ana(CSIC),Sevilla,Spain;2Department
ofBiology,SectionofEcology,UniversityofTurku,Turku,Finland;3SwissOrnithologicalInstitute, Sempach,Switzerlandand
4DepartmentofEvolution,SystematicsandEcology,TheHebrewUniversityofJerusalem,Jerusalem, Israel
Population fragmentationisawidespreadphenomenon usuallyassociatedwith humanactivity.Asaresult ofhabitat transformation,thephilopatric andsteppe-specialistLesser KestrelFalconaumanni underwentaseverepopulation decline during thelastcenturythatincreasedpopulation fragmentationthroughoutitsbreedingrange.In contrast,the ubiquitousEurasianKestrelFalcotinnunculusdidnotsuffer suchadverseeffects,itsbreedingrangestillremainingrather continuous.Usingmicrosatellites,wetestedtheeffectsof populationfragmentationonlarge-scalespatialpatternsof geneticdifferentiation anddiversitybycomparing thesetwo sympatricandphylogeneticallyrelatedspecies.Ourresults suggestthathabitatfragmentation hasincreasedgenetic differentiationbetweenLesserKestrelpopulations,following
anisolation-by-distance pattern,whilethepopulation of EurasianKestrelsispanmictic.Contrarytoexpectations, wedidnotdetectsignificantevidence ofreducedgenetic variationorincreasedinbreeding inLesserKestrels. Althoughthisstudyreportsgeneticdifferentiation ina speciesthathaspotentialforlong-distance dispersalbut philopatry-limited geneflow,largeenougheffectivepopula- tionsizesandmigrationmayhavebeensufficientto mitigate geneticdepauperation. Aseriousreductionofgenetic diversityinLesserKestrelswould,therefore, onlybe expectedafterseverepopulation bottlenecksfollowing extremegeographicisolation.
Keywords:geneticdiversity;habitatfragmentation;geneticstructure;steppelandbirds;dispersal;geneflow
Introduction
Humanactivities transformthenaturalhabitatsofmany species. Populationfragmentationoften leads tooverall reductionsinpopulation sizesand diminishesconnec- tivity amonghabitatpatches. Althoughpopulation fragmentationincreases extinctionrisksbecause of deterministic and stochasticeffectsondemographic parameters,restrictedgene flow may jeopardizelong- term persistence ofpopulations due toinbreeding depression and loss of genetic diversity. Both demo- graphicand geneticimpactsofpopulationfragmentation are believedtodependonthe number,size and spatial distributionofpopulations aswellasontimesince fragmentation.In this regard,dispersaland associated gene flowappearsoneofthemost critical factors influencing thegenetic structure and demographyof fragmentedpopulations(forexample,YoungandClarke,
2000; Frankham et al., 2002). However, restricted gene flow and the subsequent emergence of genetic
Correspondence:Dr M Alcaide,Estacio´nBiolo´gicadeDon˜ana(CSIC), Avda.MaLuisas/n,41013Sevilla,Spain.
E-mail:
Received 23 April 2008;revised2 September2008;accepted12
September2008;publishedonline 15October 2008
structuringisnot only the result ofphysicalbarriersor anatomicalimpedimentstolong-distance movement. Natal and breedingphilopatry(that is,the tendencyof individuals tobreed closetotheir birthplaceortheir previous breeding territory) is expected to enhance theeffectsofhabitatfragmentation (forexample, Greenwood,1980). Geneticdifferentiationamongfrag- ments ishence expectedtobeinverselycorrelatedwith thedispersalability ofthespecies.
In spite of all these considerations, there is not necessarily a direct association between the spatial
distributionofpopulationsand the spatial distribution
ofgenetic diversity(forexample,Dannewitzetal.,2005; Jones et al., 2007; Koopman et al., 2007). Combined
demographic and genetic investigations are therefore being encouraged to rigorously evaluate the conse-
quencesofpopulationfragmentation(for example, Koenig and Dickinson,2004).Inthisrespect, elucidating
the demographicand ecological factors that determine thedistributionofgeneticvariationatdifferentscaleshas
become fundamentalto research in conservationand evolutionary biology. Polymorphicmolecularmarkers
and powerfulstatistical methodshave allowedthe investigation of the spatial distribution of genetic
variation in fragmented populations and provided a
measureof population connectivity.Such approaches,
combinedwith life-historyand demographic informa-
tion, have consistentlyprovidedrelevantdata tounder-
pin conservation and management initiatives aimed at preserving the genetic diversity of endangered
species (for example, Caizergues et al., 2003; Mart´ınez-Cruz et al., 2004; Hansson and Richardson,
2005;Koopmanetal.,2007).
Studies ofgenetic structureand diversityin birds of
preyareaccumulatingdue toanemergingconcern about the threatsderivedfrom populationfragmentationand
habitatalteration inthischarismaticavian group(for example,Godoy etal.,2004;Mart´ınez-Cruzetal.,2004; Helbig etal.,2005;Brownetal.,2007;Cadah´ıaetal.,2007; Hailer etal.,2007; Nittingeretal.,2007).Birdsofprey typicallyhavesmallpopulations,extendeddistributional rangesand they usuallyhave long-distance dispersal capabilities. Althoughraptorpopulationstend to be poorly structured (seereferencesabove),habitatfrag- mentationcould potentiallyincrease genetic divergence amongpopulationsand reducedpopulationsizewould initiate a loss of genetic variation. In this study, we
employedpolymorphicmicrosatellites toassess the influence ofpopulationfragmentationongenetic diver-
sity and large-scale (continental) spatial patterns of
genetic differentiation in two phylogenetically related and sympatricbirds of prey, the Lesser Kestrel Falco
naumanni and the Eurasian Kestrel Falcotinnunculus. Bothspecies breed inEurasia, acontinentalmass with a
broadtradition ofhuman-inducedlandscape transfor- mations, which have generated serious threats for
the conservationof many species (Goriupand Batten,
1990;McNeely, 1994).Althoughthe Lesser Kestrel isa
specialistfalconinhabiting steppeand pseudosteppe ecosystems(Crampand Simmons,1980),the Eurasian
Kestrelisconsideredatruly cosmopolitanfalconthatcan
liveinmost open-countryenvironments(Village, 1990). Open habitatsinEuropehave increaseddue toagricul-
ture and clear-cuttingofforests, afactthat may explain why the breedingrange ofthe EurasianKestrel has not
beendecisivelyaffected byhumanactivities. Incontrast, Lesser Kestrels have experienced a well-documented
populationdecline duringthe twentiethcenturythat is mostly explainedby humanperturbations,such as the
substitutionoftraditionalagriculturalpractices by intensive agricultureand irrigatedcrops that reduce foraginghabitats(Tella etal.,1998;Ursu´a etal.,2005). Such adramaticpopulationregressionled to the extirpationordisappearanceofthe Lesser Kestrel from several Europeancountries(Biber,1990).Itconsequently
hasapatchierdistributionalbreedingrange ascompared with its generalistcounterpart (Figure 1).In addition, long-term and extensive ringing studies of Lesser Kestrels inSpain have documentedhigh natal and breeding philopatryaswellasanegativeassociation betweeneffective dispersaland geographical distance (Negro etal.,1997; Serrano etal.,2001,2003,2008). Conversely,EurasianKestrels have shownalow philo- patryand frequenteffective long-distancedispersalsin populationsfrom Northernand WesternEurope(Korpi- ma¨ki,1988;Village, 1990;Korpima¨kietal.,2006;Vasko,
2007),althoughpreliminarydata fromaSpanishpopula-
tion suggesthigherphilopatryrates inSouthernEurope
(JAFargallo, personalcommunication).
Hence, themain questionthat thisstudywilladdress iswhetherhabitatalterationhas resultedinpopulation
Figure1Breedingdistributional ranges(grey areas) ofLesser (a) and Eurasian(b)Kestrels across theWesternPaleartic. Populations analysedinthis studyareindicatedbyblack dots. Lesser Kestrels were sampled from southwestern Spain (SWS), central-western Spain (CWS),northeasternSpain (NES),France (FRA),Italy (ITA), Israel (ISR)and Kazakhstan(KAZ).The continentalsubspeciesof the EurasianKestrel was sampledfrom SWS,CWS,NES,Switzer- land (SWI),Finland(FIN)and ISR.Inaddition,two subspeciesof theEurasian Kestrel inhabitingtheCanaryIslands(indicatedby asterisks)weresampled(FVforFalcotinnunculusdacotiaeandTFfor Falcotinnunculuscanariensis).
differentiationand lossofgenetic diversityinthehighly philopatric LesserKestrelcomparedwith thewidely distributedand highly dispersiveEurasianKestrel. The suitability of the genetic methodswe used here was tested bymeansofadditionalanalyses oftwo insular subspeciesoftheEurasianKestrel inhabitingtheCanary Islands.Weexpectedthepopulationsofthesesubspecies to hold comparably lower levels of genetic variation because ofthe well-documentedeffects ofinsularityon demographyand genetic diversity (forexample,Bollmer etal.,2005).
Materialsandmethods
Studyspeciesandpopulations
The Lesser Kestrel isa small trans-Saharianmigratory falcon whose breedingrange covers mid-latitudeand
low elevationsofEurasia (Crampand Simmons,1980). This colonial falcon originally occupied small cliffs
surroundedby naturalsteppes(Tella etal.,2004),but most pairs breed nowadaysin humanstructuressur-
roundedby traditionalagriculturalland. The Eurasian
Kestrel is a sedentaryor partiallymigratoryfalcon of
slightly larger size that is widespread in Eurasia, normally showing a territorial breeding behaviour
(Crampand Simmons,1980).In Europe,the estimated
populationsizeofLesser Kestrels isabout 25000–42000 breedingpairs,whereasthatofEurasianKestrelsisabout
300000–500000 breeding pairs. We analysedbreeding
populationsoftheLesserKestrel insouthwesternSpain,
central-westernSpain, northeasternSpain, France, Italy,
Greece and Israel (seeFigure 1a).Thecontinental subspecies of the Eurasian Kestrel (Falco tinnunculus
tinnunculus) wassampledinsouthwesternSpain,central- westernSpain, northeasternSpain, Switzerland, Finland
and Israel (seeFigure 1b).Twoinsularsubspeciesofthe
Eurasian Kestrel inhabiting the Canary Islands, Falco
tinnunculus canariensisand Falcotinnunculus dacotiae(see
Figure 1b),were also investigatedtoprovidecompara-
tive data. Estimatedpopulationsizes are about 400–500 breeding pairs for F. t. dacotiae and less than 4000
breedingpairs forF.t.canariensis(Madron˜oetal.,2004). The majority of sampled individuals (490%) were
nestlings, and we only analysed one individualper brood to minimize problems associated with close
relatedness.Extra-pairpaternityinLesser and Eurasian
Kestrels has showntoberare (below 7.5%ofnestlings,
see Korpima¨kietal.(1996)and Alcaide etal.(2005)for details), and thus, theprobabilityforadultmales toraise theirownoffspringishigh.Estimatedpopulationsizesof thegeographicallydistinctpopulationsofLesserKestrels investigatedin this studyare shownin Table 1. The numberofLesserand EurasianKestrels sampledateach location isshownin3and 4,respectively.
DNAisolationandmicrosatellitegenotyping
About 100ml of blood preserved in 96% ethanol or growingfeathersthat were pulledfromthebirds’dorsal plumagewere digestedbyincubationwith proteinaseK
for at least 3h. DNA purificationwas carriedout by
using 5M LiCl organic extractionmethodwith chloro- form/isoamylicalcohol (24:1)and furtherDNA precipi-
tation using absolute ethanol. Pellets obtained were driedand washedtwice with 70% ethanol,and later
storedat —201C in 0.1ml of TE buffer. We amplified seven microsatellitesthat were isolatedoriginallyinthe
peregrinefalcon Falcoperegrinus by Nesje etal.(2000) (Fp5, Fp13, Fp31, Fp46-1, Fp79-4, Fp89 and Fp107). In
addition,wedesignedtwo setsofprimersflanking two microsatellitesequences also isolatedin the peregrine
falcon that were availablein GenBank (AF448412and
AF448411, respectively). Locus Cl347 was amplified
using primers Cl347Fw: tgtgtgtgtaaggttgccaaaand
Cl347Rv:cgttctcaacatgccagttt.Locus Cl58was amplified
using primers Cl58Fw: tgtgtctcagtggggaaaaaand
Cl58Rv: tgctttggtgctgaagaaac.For each locus, the PCR
was carriedout in a PTC-100 Programmable Thermal
Controller(MJResearch Inc.,Waltham,MA,USA)using
thefollowingPCRprofile:35cyclesof40sat941C,40sat
Table1EstimatedpopulationsizesofLesser Kestrels sampledfor thisstudy
551C,40sat721Cand finally, 4min at721C.Each11ml
reaction contained 0.2U of Taq polymerase (Bioline,
London,UK), 1xPCR manufacturer-supplied buffer,
1.5mM MgCl2, 0.02%gelatine(AmershamLifeSciences, Buckinghamshire,UK),0.12mM ofeachdNTP, 5pmol of eachprimerand, approximately,10ngofgenomicDNA. Forwardprimers were50-endlabelledwithHEX,NEDor
6-FAM.Amplified fragmentswere resolvedonanABI Prism 3100 Genetic Analyser (Applied Biosystems, Foster City,CA,USA).
Geneticanalyses
Polymorphism statisticsat each microsatellitemarker
(that is, the numberof alleles and range size of the
amplified fragments)were calculatedusing thepro- grammeGenetix 4.04(Belkhir et al., 1996–2004).Con-
formity toHardy–Weinberg equilibriumwasanalysed throughGENEPOP (Raymondand Rousset, 1995),using
a single locus and a global multilocustest for hetero- zygositydeficit orexcess bythe MarkovChain Method
(Raymondand Rousset, 1995).
WeemployedthesoftwareSTRUCTURE2.2(Pritchard
etal.,2000)totestforthepresenceofgeneticallydistinct clusterswithinour studysystem. We did not use any
prior informationabout thegeographic origin ofthe individuals,and we assumedcorrelatedallele frequen-
ciesand theadmixturemodel. Tensimulations were performedforeach ofthe Kvalues rangingfrom 1to6
(that is, the number of putatively different genetic
clusters), and probability values of the data, that is ln Pr(X/K), were plotted. Values of K¼1 indicate a
geneticallyuniform population, while values of K¼2 and soonindicatethe existence ofgeneticallydifferent
arrays of individuals. Analyseswere carriedout with
100000iterations,followingaburn-inperiodof10000
iterations. Nonetheless, testing fordifferencesinallele frequenciesbetweengeographicallydistinctpopulations
may bemore useful than clusteringanalysesperformed in STRUCTURE when genetic differentiation is weak
(forexample,Latch etal.,2006)oraffected byisolation by distance (see software documentation in
pritch.bsd.uchicago.edu/software/structure22/readme. pdf). Thus, we employed the programme GENETIX
4.04to calculate FST values betweengroupsof indivi- duals sampled from different locations of the Lesser Kestrel breedingdistribution.Althoughthe distribution range oftheEurasianKestrelisrelativelycontinuous,we alsocalculatedFST values between distantsampled locations tocontrastFST pair-wisevalues with STRUC- TUREresults.ThesignificanceofFST pair-wisecompar- isons was given by a P-value calculated using 10000 randompermutation teststhatwasfurtheradjusted accordingtosequentialBonferronicorrectionsformulti- ple tests (Rice,1989).Isolation bydistancewas investi-
gated through Mantel tests based on the traditional
Location Code Populationsize(breedingpairs)
Spanishcorearea SWSand CWS 12000–19000
EbroValley NES 1000
France FRA o100
Italy ITA 3640–3840
Greece GRE 2000–3480
Israel ISR o1000
Data were taken from BirdLifeInternational(2007),Prugnolleetal. (2003)and Liven-Schulmanetal.(2004).SeeFigure 1forgeographic locations.
FST/1—FST approach.Weintroducedinthe programme GENETIXamatrix containing values ofgenetic differ- entiationbetweeneachpairofsampledpopulations(that is, FST/1—FST values representedin the yaxis) plus a matrix containing the geographical distance in kilo- metres betweeneach pair ofsampledlocations (repre- sentedinthexaxis).Geographical distanceswere calculatedaccordingto a straightline connectingthe geometricalcentre ofeach pair ofsampledpopulations. Calculationswere accomplishedbyusing ascaled map
and aruler. Thesignificanceofthe correlationbetween
genetic differentiation and geographicaldistancewas
tested in GENETIX 4.04througha P-valuecalculated using 10000permutations.
Allelic richness, average observed heterozygosities and the inbreeding coefficient FIS among groups of samplesencompassingindividuals from differentspe- ciesorsubspecieswere comparedusing thepermutation test (N¼10000)implementedinFSTAT(Goudet,2001). Theallelicrichnessestimate,which iscalculated from randompermutationsofaminimumsharednumberof individualsbetweengroups,isespeciallyuseful inthis
study ashighly polymorphiclocisuch asFp79-4may decisivelybias estimatesofgenetic diversityinrelation to samplesize. The non-parametricWilcoxon test was also employedtodetect significantdifferencesbetween sampledlocations inpolymorphismstatisticsobtainedat eachlocus(that is,allelicrichnessand averageobserved heterozygosities).Finally, microsatellitediversityateach pairoflocations,measuredasthemeannumberofalleles per individual,was comparedusing Student’st-tests.
Results
Hardy–Weinbergequilibriumandgeneticdiversity Overall,103alleleswere found in320LesserKestrels, 75 alleles in128mainlandEurasianKestrels and 46alleles in28island EurasianKestrels (seeTable2).LocusFp107 departed significantlyfrom Hardy–Weinberg expecta- tions, showingheterozygositydeficits in most popula- tionsthat areprobablyexplainedbythepresenceofnull alleles (seealsoAlcaide etal.,2005).Asnull alleles may violate several assumptionsofthe genetic methodswe intendedtoapply,locusFp107wasremovedfromfurther analysis.MainlandpopulationsfrombothKestrelspecies fitted to Hardy–Weinberg expectationsafter excluding this locus. Wefound,incontrast,statisticallysignificant heterozygositydeficits, evenafterBonferronicorrections
for multiple tests, in the smallestinsular population
correspondingtoF.t.dacotiae.
Populationdifferentiation
InLesserKestrels, theBayesian analysis ofpopulation structureexcludingany apriori informationabout the
origin ofindividualsindicatedpanmixia(that is,K¼1,
seeFigure2) asthemostlikelyscenario. Nevertheless, traditionalestimatesofpopulationdifferentiationrelying
ondifferencesinallele frequencies revealedweak (FSTo0.055) but significantpatternsofgenetic differen- tiation, even after Bonferroni corrections for multiple tests, when wecomparedgeographically distinct populations(Table 3).Infact,genetic divergenceacross thestudyareaconformedsignificantlytoanisolation-by- distancepattern(Figure 3).
Figure 2Bayesian clustering analysis of 320 Lesser Kestrels sampledin different regions of the WesternPaleartic. For each value of K (that is, the number of putatively different genetic clusterstested), 10simulationswere carried outtoobtain the probabilityofthedata (yaxis).
Table2NumberofallelesacrossninemicrosatellitemarkersintheLesserKestrel(Falconaumanni),theEuropeansubspecies oftheEurasian Kestrel (Falcotinnunculus tinnunculus) and the two subspeciesofthe EurasianKestrel inhabitingthe CanaryIslands(Falcotinnunculus canariensisand Falcotinnunculusdacotiae)
Locusrangesize(bp) / Falconaumanni(n¼320) / Falcot.tinnunculus
(n¼128) / Falcot.canariensis
(n¼12) / Falcot.dacotiae
(n¼16)
Fp5 / 7 / 8 / 7 / 7
99–111 / 101–115 / 101–113 / 101–113
Fp13 / 5 / 4 / 2 / 4
86–106 / 92–98 / 92–94 / 92–98
Fp31 / 8 / 7 / 3 / 2
124–142 / 128–142 / 134–138 / 134–138
Fp46-1 / 10 / 6 / 4 / 6
115–139 / 117–127 / 119–125 / 115–125
Fp79-4 / 35 / 19 / 6 / 8
125–192 / 129–154 / 137–149 / 137–152
Fp89 / 4 / 5 / 2 / 4
116–122 / 116–124 / 118–120 / 116–122
Fp107 / 17 / 17 / 5 / 5
185–231 / 195–233 / 193–221 / 193–221
Cl347 / 11 / 9 / 5 / 5
96–116 / 100–116 / 100–112 / 100–112
Fp5 / 6 / NA / NA / NA
118–123 / NA / NA / NA
Abbreviation:NA,notapplicable.
Thenumberofindividualsanalysedforeachspecies orsubspeciesisshowninparentheses.
Onthe other hand,theclusteringanalysisimplemen-
ted in STRUCTURE detected only two genetically
distinctclusterswithinEurasianKestrels (that is,K¼2)
Table3Pair-wiseFST values (above diagonal)and corresponding
P-values(belowdiagonal)betweenLesserKestrelpopulationsfrom
t heWesternPaleartic
NESCWSSWSFRAITAGREISR
NES(68) / 0.008 / 0.008 / 0.014 / 0 / 0.009 0.035CWS(76) o0.001 / 0.001 / 0.019 / 0.016 / 0.014 0.041
SWS(69)0.0012 / 0.19 / 0.023 / 0.013 / 0.013 0.038
FRA(26) / 0.0021 o0.001 / o0.001 / 0.009 / 0.041 0.034
ITA(26) / 0.56o0.001 / 0.0048 0.0664 / 0.017 0.021
GRE(21) 0.002 0.0026 0.002 0.001 0.005 0.054
ISR(34) o0.001 o0.001 o0.001 0.001 0.006 o0.001
Sample sizes at each location are indicatedin parentheses. Significant values after Bonferroni corrections for multiple tests are outlinedinbold. Non-Bonferroni-correctedP-valuesare given below thediagonal.SeeFigure 1forgeographicallocations.
that distinguishedthe mainlandsubspeciesagainstthe
two insular subspecies.This finding agrees with the
comparablyhigh and statisticallysignificantpair-wise FST values reported betweenEurasia and the Canary Islands (FST40.075,allBonferroni-correctedP-values o0.05;Table4). Conversely,therewasnoevidencefor genetic subdivision within Eurasia, as none of the pair-wise FST values were significantlydifferentfrom zero (FSTo0.015, all non-Bonferroni-corrected P-values
40.05), or within the Canarian Archipelago (FST¼
—0.018, P¼0.87) (see Table 5). Contrary to Lesser
Kestrels, our set of genetic markers did not reveal
significant evidenceofisolationbydistanceinthe mainlandsubspeciesofthe EurasianKestrel (Figure 3).
Tocompare data frombothspecies, weperformeda generalized linear model with FST as the response variableand species identityand Euclidean distance betweenthepopulationsasindependentvariables.After conservatively adjustingthedenominatordegreesof freedom to compensate for the non-independence
FST
1-FST
0.06
0.05
0.04
0.03
0.02
0.01
0.00
-0.01
5.65.86.06.26.46.66.87.07.27.47.67.88.08.28.48.6
In geographical distance (km)
Figure3Relationshipsbetweenthe extent ofgenetic differentiationand geographicaldistanceinthe Lesser Kestrel (open dots, r¼0.50,
P¼0.04)and EurasianKestrel (blackdots, r¼—0.44,P¼0.84)populationssampledacross theWesternPaleartic.
Table4Pair-wiseFST values (abovediagonal) and correspondingP-values(belowdiagonal) betweenEurasianKestrelpopulationsfromthe
WesternPalearticand theCanaryIslands
FV
0.083
0.121
0.107
0.099
0.105
0.105
—0.018
Sample sizesateach location areindicatedinparentheses.Significant values after Bonferronicorrectionsformultipletests areoutlinedin bold. Non-Bonferroni-correctedP-valuesaregiven below thediagonal.SeeFigure 1forgeographicallocations.
Table5Comparisonofaveragegenetic estimatesamonggroupsof Kestrel populationsthat wasperformedusing thepermutationtest (N¼10000)implementedintheprogrammeFSTAT
Discussion
We studied the genetic implications of habitat frag- mentation by comparing the generalist, continuously
Allelic
richness
Observed
heterozygosity
Inbreeding
coefficient(FIS)
distributedmainlandsubspeciesoftheEurasianKestrel
and the steppe-specialist, patchily distributed Lesser
Lesser Kestrel5.820.660.024
Kestrel. Our findingsindicatesimilar levels of genetic
EurasianKestrel
(Mainland) EurasianKestrel (CanaryIslands)
5.280.660.084
4.240.460.265
variationinboth thespecies, but lower levelsofgenetic diversityintwo island subspeciesofEurasianKestrels.
With respect topopulationdifferentiation,the Bayesian
clusteringmethodseparatedthe mainlandpopulation
Allelic richness was calculatedover a minimumnumber of 12 individuals.
Table6Genetic diversityacross eight microsatellitemarkersinsix geographicallydistinctpopulationsofLesser Kestrels
ofEurasianKestrels from their island counterparts. Likewise, FST analysesshowedsignificant genetic differentiationbetween,but not within,these clusters. InLesserKestrels, STRUCTUREassignedallindividuals to a single putative population.Nonetheless,the esti- mates ofpopulationdifferentiationthat made useofthe additionalinformationonthegeographicdistributionof allele frequenciesrevealedlow but significantlevels of
Allelic
richness
Averageobserved
heterozygosity
Inbreedingcoefficient
(FIS)
genetic differentiationfollowinganisolation-by-distance model.
NES6.60.630.07
CWS+SWS7.060.650.05
FRA6.020.600.04
ITA6.890.67—0.06
GRE6.880.640.01
ISR7.420.660.03
Itiscurrentlyassumedthat species thrivingwithina range of environmental conditionsare more sensitive
to habitat transformations, their distributional ranges
becomingpatchierand the risk forgenetic drift within fragments increasing(for example,Ferrer and Negro,
2004).Our empiricalapproach exemplifiesa situation
Allelicrichnessestimateswere adjustedtoaminimumsamplesize
of21individuals.SeeFigure 1forgeographicallocations.
between sampling locations (see Bailey et al., 2007), the interaction term remained significant (F1,9¼9.11, P¼0.015).
Geneticdiversity
ThepermutationtestperformedinFSTATdid notreveal statistically significant differences in genetic diversity
(allelic richnessand averageobservedheterozygosity) or inbreeding (FIS) between the Lesser Kestrel and the mainland subspecies of the Eurasian Kestrel (all two-sidedP-values40.05, Table 5).Incontrast,average observed heterozygosity was significantly lower in island than inthecontinentalsubspeciesoftheEurasian Kestrel (0.46vs0.66,two-sidedP-value¼0.009;Table5), and thedifferenceinallelicrichness wasmarginally significant (4.24 vs 5.28, two-sided P-value¼0.08;
Table 5).Furthermore,we foundstatisticallysignificant evidencesofincreased inbreeding(FIS)intheKestrel genotypes from the Canary Islands (0.265 vs 0.084, two-sidedP-value¼0.02;Table5).
Finally,pair-wiseanalyses comparinglocusbylocus failed todetect statistically significantdifferencesin genetic diversity between any of the geographically distinctpopulationsofLesserKestrels investigated(non- parametricWilcoxontest,allP-values40.05;seeTable6). Averagemicrosatellitediversityper individualwas not statisticallydifferentamongpopulations either (t-tests, allP-values40.05), exceptforacouple ofcomparisons involvingthesmallestand themost geographically isolatedpopulationfromSouthernFrance.Suchcompar- isons involvedthe less geneticallydiversepopulation (France) and two ofthe most geneticallydiverse(Italy and Israel, seeTable6)populations.
whereby genetic differentiation reflects the spatial
distributionofpopulations,which, inturn, isdelimited by habitat requirements. Thus, genetic differentiation
between Lesser Kestrel populations increases with geographicaldistance(see also Alcaide etal.,2008for
data on MHC genes). Even thoughLesser Kestrel isa long-distancemigratoryspecies, gene flow isrestricted
over short distances due to high natal and breeding philopatry(Negro etal.,1997;Serrano etal.,2001;Serrano
and Tella,2003).Elsewhere,wefound,however, alackof fine-scale patterns ofgenetic differentiationinaspatially
structured population of Lesser Kestrels located in northeasternSpain (Alcaide etal.,inpress). Thisfinding
was attributedtothefactthat populationsubdivisionat the geographicalscale studied(about 10000km2) may
not have been sufficient, given the long-distancedis- persal capabilities displayed by the species; conse-
quently,gene flow had homogenizedallele frequencies. Nonetheless,effective long-distancedispersalbyLesser
Kestrels (4100km) has rarely been documented by direct observations(Prugnolleetal.,2003;Serrano etal.,
2003;PPilard and FMart´ın, personalcommunication;D Serrano et al., unpublisheddata; M Alberdi,personal
communication),afactthat wouldbeinagreementwith theemergenceofgenetic structuringatlarge geographi-
cal scales. In contrast, it has been shown in several
Europeanpopulations of Eurasian Kestrels that natal
dispersal regularly occurs over large distances (for
example,Snow, 1968;seealsoKorpima¨ki,1988;Village,
1990; Korpima¨ki et al., 2006; Vasko, 2007). This high amplitudeofdispersal,combinedwiththelowincidence
ofhabitatfragmentationinthe EurasianKestrel, would
thereforeexplain itsgenetic uniformity.
Populationgenetics theory predictsthat reductionsin
population size and limited migration decrease local genetic variation,triggeringnegativegenetic processes
such as inbreeding depression and loss of adaptive potential (Frankham et al., 2002). Following these
predictions, recent studiesin the Lesser Kestrel have
repeatedly found weak positive correlationsbetween
fitness componenttraits and individualgenetic diversity at11polymorphicmicrosatellitemarkers(Ortego etal.,
2007b,c).However,our genetic analyses,relying on at least sixmicrosatellitespreviouslyamplifiedbyOrtego
and co-workers(Fp5, Fp13, Fp31, Fp46-1, Fp79-4 and
Fp89), have not revealed comparably low levels of
microsatellitediversityorincreasedinbreedinginLesser
Kestrels inrelationtotheputativelyoutbredsubspecies
oftheEurasianKestrel. Genetic variationatfunctionally and evolutionaryrelevantMHC loci have also shown
extraordinarylevelsofpolymorphism(4100alleles ata single locus) and heterozygositiesabove 95%in Lesser
Kestrels (Alcaide etal.,2008).
We believe that additional analyses of the pre-
bottlenecked population are needed to evaluate the degreeof genetic depauperationin the Lesser Kestrel.
Inanycase,itappearsincautious toassumethat the populationdeclineexperiencedbythisspeciesislikelyto
have translated into reduced levels of contemporary genetic variationand increasedinbreeding.Forinstance,
Brown et al. (2007) have recently failed to detect signaturesof a genetic bottleneckin peregrine falcons
after adevastatingdecline inthemid-twentiethcentury due to organochlorine contaminants. Similarly, some
Lesser Kestrel populationshave been knowntoexperi- encedemographicgrowth,either naturally(forexample,
Tellaetal.,1998; Ortego etal.,2007a)orbymeansof reintroduction or supplementation programmes (for
example,Pomarol,1993).Yeteven in the bottlenecked and geographicallyisolatedpopulation from Southern
France, from where wereportthelowest levelsof microsatellite polymorphism (Table 6), there is no