OIKOS 103: 162–174, 2003

Dispersal of the lycaenid Plebejus argus in response to patches ofits mutualist ant Lasius niger

Adrian S. Seymour, David Gutierrez and Diego Jordano

Seymour, A. S., Gutierrez, D. and Jordano, D. 2003. Dispersal of the lycaenidPlebejus argus in response to patches of its mutualist ant Lasius niger. – Oikos 103:162–174.

We examined the movement rates of individuals of an obligate myrmecophilousbutterfly, Plebejus argus (Lycaenidae: Lepidoptera), in relation to a patch of itsmutualist ant, Lasius niger. Marked male and female P. argus from local (resident),fragmented and continuous populations were released into and away from (�100m)a patch of L. niger, and subsequently searched for in a 500×500 m area. Recapturesof males and females from all populations were strongly associated with the targetpatch of mutualist ants. First day recapture displacements were significantly higherfor releases away from the target patch for males, but not for females. Mutualist antpresence was shown to be more important than nectar source density in determiningpersistence of P. argus in release squares. There was no significant difference indisplacement distances or recapture frequencies (standardised for search effort) in thetarget patch of individuals from local (resident of target patch), fragmented andcontinuous populations. The recapture frequency in the target patch of males fromthe local population released outside the target patch was greater than expected froma null model of random straight line movement, suggesting non-random (systematicor orientated) search behaviour. The number of males remaining in the target patchfollowing release into the target patch was greater than expected from a simplediffusion model, suggesting a behavioural response to patch boundaries or other cuesrelated to the patch.

A. S. Seymour, D. Gutierrez and D. Jordano, Di�ision de Ecologıa, Uni�. de Cordoba,Colonia San Jose No. 3, Campus Rabanales, Cordoba, ES-14071, Spain (aseymour–uk@yahoo.co.uk). Present address for ASS: 10 Augustine Road, London, W14 0HZUK. Present address for DG: Area de Biodi�ersidad y Conser�acion, Escuela Superiorde Ciencias Experimentales y Tecnologıa, Uni�. Rey Juan Carlos, Tulipan s/n,Mostoles, Madrid, ES-28933, Spain.

Because of increasing habitat loss and fragmentation,there is a growing interest in the role of ecologicalprocesses operating at the landscape scale on popula-tion persistence (Edwards et al. 1994). Populations oc-curring in fragmented landscapes are usually smallerand more isolated, which makes them more vulnerableto local extinction events due to stochastic and deter-ministic processes. The long-term persistence of localpopulations based solely on local births and deaths isstrongly limited, and the movement of individualsamong populations has a crucial role in rescuing extantlocal populations or (re)colonising empty habitat

patches (metapopulation concept: Hanski 1999). Never-theless, for most species, the local components (birthand death) of their population growth rates are stillmuch better understood than their regional components(immigration and emigration). This is partly due topractical problems associated with collecting field dataon dispersing stages at large spatial scales.

Many butterfly populations with patchy distribu-tions, including many species of conservation concern,have been shown to be structured as metapopulations,and their local persistence mostly depends on the dy-namics at a regional scale (Harrison et al. 1988,

Accepted 29 March 2003

Copyright © OIKOS 2003ISSN 0030-1299

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Thomas and Jones 1993, Hanski et al. 1994, Thomasand Hanski 1997, Gutierrez et al. 1999, Baguette et al.2000). In most cases, suitable habitat patches weredenoted by the presence of larval host plants or amarked variation in plant quality, surrounded by amatrix habitat where breeding cannot occur, formingdistinct patch boundaries.

A number of empirical studies have shown that thestructure of patch boundaries can influence movementbehaviour: low-contrast boundaries tend to be morepermeable than high-contrast boundaries associatedwith large differences in habitat structure (Kuussaari etal. 1996, Schultz 1998, Haddad 1999, Ries andDebinski 2001). The permeability of patch boundariescan have important effects on patch emigration rates(Stamps et al. 1987), thus altering the rate at whichindividuals are lost from patches into the surroundingmatrix, and the number of migrants available forcolonisation. In addition, a number of studies haveexamined how butterflies locate patches of suitablehabitat, a behaviour that may have important implica-tions for patch colonisation rates (Harrison 1989, Con-radt et al. 2000, Schultz and Crone 2001). These studiesshowed that orientation to patches may occur within alimited distance to the patch (usually �50 m), and thatat least in one case, a systematic search strategy may beemployed to locate habitat patches (Conradt et al.2000). All these studies have concentrated on systemswith distinct habitat patch boundaries, easily dis-cernible to the human eye. However, many Lycaenids(and other mutualist species) occur in patches deter-mined by the distribution of their mutualist species(Smiley et al. 1988, Jordano et al. 1992, Costa et al.1996, Seufert and Fiedler 1996), that may differ verylittle in vegetation structure from the surrounding ma-trix habitat. Although there has been considerable workon how Lycaenids select oviposition sites with respectto host ants (Atsatt 1981, Pierce and Elgar 1985, Pierceand Easteal 1986, van Dyck et al. 2000), very little isknown about the larger scale movements related toemigration from and immigration to patches of mutual-ist ants.

In this paper we report a set of release experimentswith Plebejus argus, one of the relatively few obligatemyrmecophilous Lycaenids in Europe. Releases werecarried out in and far away (�100 m) from mutualistant patches in Donana National Park at the southernlimit of the European range of P. argus. The host plantHalimium halimifolium is widely distributed throughoutthe xerophytic scrubland of the park, but the distribu-tion of P. argus is locally correlated with that of its hostant, Lasius niger (Jordano et al. 1992). Due to hetero-geneity in L. niger distribution, the distribution of P.argus is almost continuous along the lower elevationeastern and southern parts of the xerophytic scrubland,and fragmented in the higher and drier areas in westernparts (unpubl.). The limits of these population frag-

ments are not associated with any distinct change invegetation type. Landscape structure is predicted tohave an important effect on the evolution of movementbehaviour by changing, for example, the relative costsand benefits of different mate and oviposition sitesearching behaviours (van Dyck and Matthysen 1999),as well as dispersal behaviour per se (Olivieri et al.1995, Heino and Hanski 2001). Our study system,which consists of both fragmented and relatively con-tinuous distributions of P. argus provided us the oppor-tunity to test these predictions.

The objectives of this study were: 1) to determinewhether P. argus from fragmented and continuous dis-tributions show the same dispersal behaviour, 2) todetermine whether P. argus actively selects habitat con-taining host-ants, 3) to determine whether P. argus usesnon-random search strategies to locate patches of hostant, and 4) to determine whether the limits of host-antpatches act as semi-permeable boundaries for P. argus.

Methods

Site and study species

Donana National Park is located on the southwesterncoast of Spain, and covers approximately 50,000 ha,including fixed dunes, mobile dunes, and marshes. Thesandy soil has a very low field capacity and the vegeta-tion on the fixed dunes is strongly influenced by thewater table depth. The driest areas are covered byscrubland dominated by H. halimifolium, Rosmarinusofficinalis and other species of Cistaceae, whereas thewettest sites are covered by dense heathland with Ericascoparia, Ulex minor and Rubus spp. Further details onDonana are given by Fernandez-Delgado (1997).

P. argus is the most abundant butterfly in Donana,and is univoltine, flying from late April to early July.Its larvae are monophagous on the most abundant andwidespread shrub in the area, H. halimifolium, whichoccurs in nearly all vegetation types in Donana. How-ever, egg placement by P. argus is clearly biased towardthose host plants with L. niger nests at the base; thisrelationship becomes even closer for the larval andpupal stages, which are mostly restricted to those plantsbearing L. niger nests (Rodrıguez et al. 1991, Jordanoet al. 1992).

Collection and release sites

From an intensive study of 50 one-hectare plots (un-publ.), we found that P. argus abundance was closelycorrelated with L. niger nest density, which could bepredicted by three variables readily measurable frommaps and aerial photographs (elevation, presence ofheathland vegetation, and nothing in the one hectare

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plot). In order to determine the spatial structure ofDonana’s P. argus population, we extended the modelto each 1 hectare quadrat in Donana’s xerophyticscrubland using a GIS (unpubl.). The resulting mapof P. argus abundance showed that at the 1 hectareresolution, P. argus distribution was fragmented inwestern parts of the Park, and continuous in thelower elevation areas to the south and east (Fig. 1).A validation of the predictive model with an indepen-dent data set indicated that the map provided an ade-quate description of P. argus distribution. A fulldescription of the development and test of the predic-tive map of P. argus abundance will be presentedelsewhere. We used the map to identify suitable re-lease sites and areas for collecting butterflies.

We selected two release points, one situated withina relatively isolated patch of P. argus (‘target’ patch),and the other located approximately 100 m from theedge of the same patch (Fig. 2). In each of the 1

hectare UTM squares centred around the releasepoints we estimated L. niger nest and nectar source(flower) density. We measured L. niger density bysearching for ant nests along twelve 10×2 m bandsspaced evenly throughout the square. In Donana, L.niger only makes nests around the bases of bushesdue to the instability of the sandy soil, so searcheswere restricted to H. halimifolium bushes. We esti-mated the density of nectar sources by counting flow-ering plants within twenty 2×2 m quadrats randomlyplaced within each quadrant (50×50 m) of the re-lease square (i.e. 80 quadrats per release square).

We selected three butterfly collection areas (Fig. 1).The first collection area was the P. argus patch asso-ciated with the release sites (‘local’ population). Thesecond collection area consisted of distinct patches ofP. argus separated from the release patch by at least400 m (‘fragmented’ population). The distribution ofP. argus in the third collection area was continuous,which we confirmed by surveying all one hectareUTM cells within a square kilometre covering thearea (‘continuous’ population).

Finally, we estimated the limits of L. niger distribu-tion around the target patch using a series of five 100m transects laid perpendicular to the edge of theheathland vegetation found at the centre of the targetpatch (Fig. 2). In each ten metre segment of thesetransects we searched for L. niger nests at the basesof the first 10 plants encountered within two metresof the transect.

Fig. 1. Map showing the butterfly collection sites (white cir-cles) in fragmented and continuous landscapes. Estimated L.niger density (proportion of H. halimifolium plants with antnests at the base) is shown at a 1 hectare resolution from aGLM habitat model including elevation, presence of heathlandand nothing as predictors. Heathland vegetation patches arealso shown. Squares marked ‘‘A’’ and ‘‘B’’ denote the releasesites shown in Fig. 2.

Fig. 2. Map of release sites in xerophytic scrubland habitat.Small squares labelled A and B show the 1 hectare UTMrelease squares (see also Fig. 1). The larger squares show the500×500 m search areas.

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Capture, release and recapture protocol

A total of six releases of around 200 butterflies perrelease were carried out between 31st of May and the7th of June 2001. Releases were made during stableatmospheric conditions characterised by clear skies andlittle or no wind. The first three releases were madeoutside the target patch (site A) using butterflies drawnfrom 1) the local population (residents of the targetpatch), 2) the fragmented population and 3) the contin-uous population. The final three releases (one for eachpopulation) were carried out inside the target patch(site B). For each release, the sex ratio of releasedindividuals was close to 1:1.

Butterflies from the fragmented and continuous pop-ulations were collected in equal numbers from threewell separated (�400 m) collection points within eacharea to ensure a representative sample from each area(Fig. 1). Butterflies were captured in the afternoon,usually between 3:00 pm and 6:00 pm, using a butterflynet. Captured individuals were transferred to plasticcontainers with perforated lids along with some Thymusmastichina or La�andula stoechas flowers, the favourednectar sources for P. argus in Donana. The plasticcontainers were stored in an insulated box kept coolwith ice-blocks. After collecting, each butterfly wasindividually marked, and its sex and level of wing-wearrecorded using a scale of 1 (very poor) to 4 (perfect).After being marked, butterflies were returned to theircontainers and stored in the ice-box until release. Themarked butterflies were taken to the release site aftersunset, and decanted onto host plants close to thecentre of the release square. The density of releasedindividuals was comparable to natural night-time densi-ties of P. argus, in which tens of individuals can befound roosting on single bushes.

We searched for marked butterflies the following daywithin a 500×500 m search area centred around therelease square (Fig. 1). Searches began at approxi-mately 11:00 am. In Donana, P. argus starts to becomeactive between 8:00 am and 9:00 am, so the markedbutterflies had plenty of time to disperse before webegan recapturing individuals. The search area wascovered systematically, starting with 45 minutes of re-capture effort by three recorders within the releasesquare. Following search in the release square, therecorders split up, each searching eight 1 hectare cellswithin the search area for approximately 10 minuteseach (i.e. 24 cells in total). When this was completed,the three recorders converged to the target patch wherethe majority of butterflies were located, and searchedfor marked butterflies for an additional hour. Theposition of marked butterflies was obtained using hand-held GPS receivers (GPS 12XL, Garmin Corp.), andtheir sex and condition noted.

Data analysis

Effect of source population structure on mo�ementWe carried out three separate analyses comparing dif-ferent aspects of movement behaviour of butterfliescollected from local, fragmented and continuous popu-lations. In the first analysis, we tested for effects of sex,population (i.e. local, fragmented and continuous) andrelease site on first day recapture displacements. Due tothe heteroscedasticity and non-normality of the data’serror structure it was not possible to carry out astandard analysis of variance. Instead, we fitted a gen-eral linear model to our data using the type 3 analysisof the GENMOD procedure in the SAS statisticalsoftware package (SAS Institute Inc., 1999). The type 3analysis is similar to using type III sum of squares in anormal analysis of variance, except that likelihood ra-tios are used instead of sum of squares. This methodwas selected because the results do not depend on theorder in which terms are specified in the model. Thedisplacement data were modelled assuming a gammaerror distribution, reflecting the zero lower limit andright hand skew of the observed data.

In the second analysis, we compared the relative‘immigration efficiency’ of butterflies from local, frag-mented and continuous populations. We measured rela-tive immigration efficiency as the number of butterfliesreleased outside the target patch that were recapturedin the target patch the following day. This measureincorporates the rate at which butterflies move throughmatrix habitat, the ability to locate patches, the ten-dency to enter and remain in patches, and survival. Solong as recapture effort and weather conditions remainequal between releases, it is possible to test for associa-tions between recapture in the target patch and popula-tion and sex using log-linear analysis. Recaptures wereclassified by recapture (captured in target patch, NOTcaptured in target patch), sex (male and female), andpopulation (local, fragmented, continuous). Only firstday recaptures were included in these analyses in orderto standardise capture effort. We identified associationsbetween recapture and the other variables using thebackward elimination method in SPSS’s log-linearmodel selection procedure (SPSS Inc., 1998).

In the third analysis, we compared ‘emigration ten-dency’ of butterflies from local, fragmented and contin-uous populations. We measured relative emigrationtendency of butterflies released within the target patch,as the number of recaptures in the target patch thefollowing day after a standard search effort. This mea-sure incorporates the rate at which butterflies movethrough suitable habitat, the tendency to move out ofthe habitat patch, and survival. We tested for associa-tions between recapture frequencies in the target patch,population and sex using a log-linear analysis in exactlythe same way as described in the preceding analysis.

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Habitat selectionTwo analyses were carried out to determine whetherP. argus selects habitats associated with L. niger ants.Firstly, we tested whether recaptures within the entiresearch area were distributed independently of habitattype (i.e. patch and matrix) using a chi-squared test.Expected frequencies, E, were calculated using the fol-lowing equation:

E(target patch or matrix)

=Ni×Areatarget patch or matrix/Areatotal

where Ni is the total number of individuals recapturedfrom release i, Areatarget patch and Areamatrix are thearea of the target patch and matrix habitat enclosedwithin the search area respectively, and Areatotal is thetotal search area equal to 500×500 m2 minus thearea of impenetrable heathland vegetation in thesearch square. This analysis was carried out separatelyfor recaptures made on the first day after release, andrecaptures made on subsequent days after releasewhen individuals had more time to disperse.

Secondly, we compared recapture frequencies withinthe two release squares for which we had measures ofnectar source density. Only first day recaptures wereincluded in the analysis in order to standardise forcapture effort. The frequency data were classified byrelease site (A and B), sex (male and female), ‘popula-tion’ (local, fragmented, continuous), and recapture(captured in release square, NOT captured in releasesquare). We identified associations between recaptureand the other variables using the backward elimina-tion method in SPSS’s log-linear model selection pro-cedure (SPSS Inc., 1998).

Orientation to patchesTo determine whether P. argus released in matrixhabitat were capable of locating the target patch us-ing an efficient non-random search strategy, we com-pared the observed frequency of recaptures in thetarget patch, with the expected frequency based on anull model of random search (Harrison 1989). Thenull model assumed (i) non-orientated linear move-ment from the release site, (ii) individuals entering thetarget patch remained in the target patch, (iii) nomortality between release and recapture, (iv) recaptureof all individuals entering the target patch, and (v)released individuals had enough time to reach theborder of the target patch. The expected frequency Eof individuals released from point A that are recap-tured in the target patch under this null model can becalculated as:

E=N0�/360

where N0 is the number of butterflies released at pointA, and � is the angle subtended by the target patch.The assumptions of straight-line movement, no mortal-ity and recapture of all individuals reaching the targetpatch are clearly unrealistic. However, by includingthem we make the test more conservative (i.e. harder toreject the null hypothesis). Observed and expected fre-quencies of males and females in and out of the targetpatch from each population were compared using chi-square tests.

Patch boundary permeabilityIf a patch boundary were completely permeable, andbutterflies moved randomly within the patch, the rateof loss of butterflies from the patch could be describedby a diffusion process. However, if the patchboundary acts as a semi-permeable barrier to butterflymovement, or if butterflies show other mechanisms formaintaining their position within a patch (e.g. re-ori-entation towards the centre of a patch or turningbias), we would expect to find more butterflies in thepatch than predicted by a diffusion model. In thiscase, it was not appropriate to use simple diffusionmodels parameterised with the recapture data to pre-dict recapture frequency within the target patch, be-cause of the habitat heterogeneity in our system(Turchin 1998). Instead, we used an individually basedsimulation model of P. argus movement to predict theexpected number of individuals remaining in the targetpatch following release in the target patch. The modelwas written in FORTRAN 77 on a UNIX mainframe,and simulated movement by using move distances,turn angles and displacement rates randomly drawnfrom empirical distributions to calculate new positionsand update time elapsed (Siniff and Jessen 1969). Theempirical distributions of movement data were ob-tained by tracking a sample of 67 males and 34 fe-males in habitat with L. niger using the methods ofTurchin et al. (1991). We simulated dispersal for eachof the butterflies released into the target patch thatwere subsequently recaptured. In order to make thepositions of simulated recaptures comparable withthose of observed recaptures, the simulated butterfliesmust be allowed to disperse for the same time periodas experimentally released butterflies (i.e. time betweenrelease and recapture). We measured how much timeP. argus dedicates to flight throughout the day, byfollowing a sample of ten males and ten females fortwo minutes in each hour between 8:00 am and 8:00pm, following the method of Schultz (1998). Thesedata allowed us to estimate the time dedicated to flightbetween release and recapture. In order to determinethe expected proportion of released individuals recap-tured in the target patch we repeated each individualsimulation 500 times. The expected frequency of re-captures in the target patch was calculated as:

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Table 1. Characteristics of the 1 hectare release sites. Site names correspond to those in Fig. 2.

Mean nectar source density (plants m−2�s.d.)Release site L. niger density (% hostplants with nests)

La�andula stoechas Thymus mastichina

A 0.362�0.094 (n=80)0% (n=96) 0.037�0.021 (n=80)B 16% (n=82) 0.012�0.012 (n=80) 0.087�0.036 (n=80)

E=N0×Nsimtarget/Nsimreleased

where N0 is the number of butterflies released experi-mentally in the target patch, Nsimtarget is the totalnumber of all simulated paths terminating in the targetpatch, and Nsimreleased is the total number of simula-tions (i.e. N0×500).

The target patch also has an internal boundary withthe heathland vegetation (Fig. 2), which may itself beselectively permeable and alter expected recapture fre-quencies in the target patch. In order to control forthis, the simulations were run again, this time assumingthat the internal boundary was impermeable. Boundaryimpermeability was modelled in the following way: ifthe co-ordinates of a simulated butterfly movementpath fell inside the area of heathland vegetation, thennew co-ordinates were calculated from a newly sampledmove length and turn angle. This was repeated until thenew co-ordinates fell outside the area of heathlandvegetation.

Results

Total recapture rates ranged from 20% to 39% ofbutterflies released. The majority of butterflies werecaught on the first day after releases, though significantnumbers of captures were made on following days. Thelongest time between release and recapture was 8 daysrecorded for a female from the first release.

Characteristics of release squares

Nectar sources were present in both release sites,whilst L. niger was absent in release square A, butpresent in B (Table 1). The flower species mostcommonly used as a nectar source by P. argus in ourstudy area were La�andula stoechas and Thymus mas-tichina. There was no significant difference in the den-sity of L. stoechas between release squares (t=1.0,n=160, n.s.), but the density of T. mastichina wassignificantly greater in release square A (t=2.7, n=160, P�0.01). The density of L. niger nests fell rapidlywith distance from the heathland vegetation at thecentre of the target patch, always dropping to unde-tectable levels beyond 80m from the heathland vegeta-tion (Fig. 3).

Effect of source population structure onmovement

DisplacementsMedian displacements of recaptures made on the firstday after release are presented in Fig. 4. The generallinear model of first day displacements using sex, re-lease site, population and their interactions as explana-tory variables provided an adequate fit to the data(Table 2). Both sex and release site and their interactionhad a significant effect on displacements of first dayrecaptures (Table 2): males were recaptured at greaterdistances from the release sites than females, with malesmoving greater distances when released at site A (L.niger absent) than site B (L. niger present), and femalesshowing the reverse pattern (Fig. 4).

Immigration efficiency and emigration tendencyFirst day recapture rates within the target patch variedfrom 5% to 15% of individuals released. Because firstday target patch recapture frequencies were low (Table3), we collapsed the contingency table over wing-wear.The frequencies of wing-wear categories in releasedbutterflies were distributed independently of release site,population and sex (�2=17.9, d.f.=18, n.s.), so therewere no reasons a priori to expect biases due to wing-wear.

The first day recapture frequencies in the target patchof individuals released outside the target patch (relative‘immigration efficiency’) were dependent upon sex

Fig. 3. The proportion of H. halimifolium plants associatedwith L. niger nests with distance from heathland vegetation intarget patch. Proportion of plants with L. niger nests calcu-lated for each 10 m section of all five transects combined (i.e.n=50 plants for each 10 m interval).

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Fig. 4. Median displacements� inter-quartile range of firstday recaptures by population, sex and release site.

Habitat selection

Test of independence of recapture position and habitattypeThe spatial distribution of recaptures from A and B onthe first and subsequent days following release arepresented in Fig. 5. Since we failed to find any differ-ences in recapture frequencies in the target patch ordisplacements between populations, for the followinganalysis we pooled data across populations. The firstday frequency of recaptures in the target patch offemales released outside the target patch (site A), waslower than expected from a random distribution ofrecaptures in the search area (Table 4), probably due totheir slow displacement rate. However, recaptures offemales released from A after the first day followingrelease showed a strong association with the targetpatch (Table 4). Recapture frequencies of females fromsite B and males from both release sites were signifi-cantly higher in the target patch than expected from arandom distribution, both on the first and subsequentdays after release (Table 4), suggesting strong selectionfor the target patch.

Comparing recaptures in release squaresIn order to determine the relative importance of nectarand L. niger availability on habitat choice by P. argus,we analysed recapture frequencies in the release squareswhere both flower density and ant abundance weremeasured. The percentage of males and females recap-tured in the two release squares after a fixed searcheffort of 135 man minutes on the first day after releaseis shown in Fig. 6. Despite the higher flower density inrelease site A, there were fewer males recaptured inrelease square A than in square B. In contrast, the firstday recapture rates of females were higher in releasesquare A than B, particularly for the ‘fragmented’population (Fig. 6). First day recapture frequencies inthe release square were cross-classified by release site,population, and sex. The log-linear model that bestdescribes the observed frequencies showed that recap-tures in the release squares were dependent upon releasesite×sex and release site×population interactions (Fitof log-linear model to data: �2=28.0, d.f.=31, n.s.).

Despite the high first day recapture frequencies offemales in release square A, only three (1.3% of releasedindividuals) were captured in the second day followingreleases, and none in subsequent days. This pattern wasreflected by males released from A, with only four(1.4% of released individuals) recaptures in the releasesquare in the second day after release. Individualsreleased into square B were recaptured in the releasesquare in relatively high frequencies up to the end ofthe experiment, four days after the first release intosquare B. For the first release into square B (localpopulation), 22% of males and 13% of females recap-tured in the release square on the first day were againrecaptured in the release square two days later.

Table 2. Likelihood ratio statistics for general linear model ofdisplacements as a function of sex, release site, population andtheir interactions assuming a gamma response distribution(Log likelihood=−1308.2, scaled model chi-squared=458.5,d.f.=269, n.s.).

Source d.f. Chi-squared P

0.8940.222Population (POP)1Release site (RS) 0.0048.38

Sex (SEX) 0.00077.431RS×POP 2 3.79 0.150

3.72POP×SEX 2 0.1561 22.40RS×SEX 0.000

RS×POP×SEX 2 2.28 0.320

(males 12%, females 2%) but not upon population(‘best’ log-linear model: log yijk=�+�RECAPTURE(i)+�SEX(j)+�POPULATION(k)+�RECAPTURE×SEX(ij); �2=6.2, d.f.=6, n.s.). However, the first day recapturefrequencies in the target patch of individuals releasedwithin the target patch (relative ‘emigration tendency’)were independent of sex and population (log-linearmodel with no interactions: log yijk=�+�RECAPTURE(i)+�SEX(j)+�POPULATION(k); �2=5.9,d.f.=7, n.s.).

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Table 3. Frequency of marked Plebejus argus cross-classified by release site, source population, sex, and recapture in the targetpatch on the first day after release. Butterflies not captured in the target patch include individuals that were not recaptured andthe those that were recaptured outside the target patch.

Population Captured in target patchRelease site Not captured in target patch

Males Females Males Females

A Local 13 1 77 46Fragmented 12 2 81 75Continuous 9 2 96 100

B Local 15 20 80 82Fragmented 17 11 88 81Continuous 21 14 79 86

Fig. 5. Recapture positions of butterflies released from site A, (a) on the first day after release, (b) on �2 days after release,and butterflies released from site B, (c) on the first day after release, and (d) on �2 days after release.

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Table 4. Observed and expected recapture frequencies in target patch in the first and following days after release. Asterisks showsignificance levels of chi-square tests with one degree of freedom (observed and expected frequencies outside target patch notshown). Fisher’s exact test was used when expected frequencies were less than 5. * P�0.025, *** P�0.0001.

Release site Sex 1 day after release �1 day after release

ExpectedObserved Expected Observed

3A Male 34*** 9 24***Female 35* 12 23***

B 7Male 53*** 29 19***Female 45*** 622 15***

Orientation to patches

Observed target patch recapture frequencies of individ-uals released outside the target patch were greater thanexpected from the null model (non-orientated straight-line search) in three cases: males from the local popula-tion, females from the local population and males fromthe fragmented population (Table 5). The differencewas significant for males from the local population(Table 5).

Patch boundary permeability

We tested whether the boundary of the target patchacted as a semi-permeable barrier to P. argus move-ment by comparing observed recapture frequencies

within the target patch with expected frequencies gener-ated by an individually based simulation model. Themovement and activity data used to parameterise themodel are presented in Fig. 7. In seven cases, theobserved recapture frequencies of individuals releasedfrom site B were greater than expected from the nullmodels (Table 6). The observed target patch recapturefrequencies of males from the local and continuouspopulations were significantly greater than expectedfrom both the null models (Table 6), showing that theyhave some kind of behavioural mechanism for main-taining their position within the target patch. The ob-served target patch recapture frequency of males fromthe fragmented population was not high enough toreject the null hypothesis for model 2 (internalboundary with heathland vegetation assumed to beimpermeable to P. argus). This may have occurredbecause there was only one day available for recapturesfollowing the release of this population before theexperiment ended. The observed recapture frequency offemales in the target patch was greater than expected inone case, but not significantly so.

Discussion

Effect of source population structure on movement

Several theoretical studies predict that habitat patchi-ness is an important factor selecting for dispersal andother movement behaviours (Olivieri et al. 1995,Olivieri and Gouyon 1997, Heino and Hanski 2001).Indeed, Hanski et al. (2002) found that the dispersalrate of female Glanville fritillary butterflies Melitaeacinxia was greater for individuals from newly estab-lished populations. However, in this study we failed tofind any differences in dispersal ability or emigrationtendency of P. argus drawn from fragmented and con-tinuous habitats. One possible explanation is that theremay be little population turnover in patches even in themore fragmented habitats, and therefore no recentlyestablished populations. In the fragmented habitat,even the smallest patches of a hectare or less supportedpopulations of P. argus numbering in their hundreds,suggesting that these populations may have a low prob-

Fig. 6. The percentage of released butterflies recaptured in the1 hectare release squares after 135 man minutes search on thefirst day after release. See text for more details.

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Table 5. Observed and expected frequencies of total recaptures in and out of the target patch of individuals released from A.Expected frequencies obtained from a null model of non-orientated straight line movement. Chi-squared values only calculatedwhen observed recapture frequencies in the target patch are greater than expected frequencies (i.e. 1-tailed hypothesis; *P�0.025).

Population Sex Capture position Observed Expected �2

Local M Target patch 25 16.7 5.08*Matrix 64 72.3

0.12F Target patch 12 10.875Matrix 92 92.125

Fragmented M Target patch 21 16.7 1.36Matrix 68 72.3

F Target patch 9 15.1875Matrix 72 65.8125

Continuous M Target patch 11 19.3125Matrix 92 83.6875

F Target patch 6 18.9375Matrix 95 82.0625

ability of extinction. High-density populations of P.argus in habitat patches in British metapopulationswere ultimately driven to deterministic extinction byplant succession leading to unfavourable microclimatesfor the host ant (Thomas and Harrison 1992). Similarprocesses are unlikely to occur in Donana, because theabundance of L. niger appears to be unrelated tovegetation characteristics (unpubl.). However, smallpatches in high elevation areas may be particularlysusceptible to drying in years of low rainfall, thus

stimulating the loss (by emigration or mortality) of thehost ant L. niger which require a certain level of soilhumidity to maintain their nests. This would lead to thedeterministic extinction of even relatively high-densitypatches of P. argus. High population densities in smallhabitat fragments, and the butterflies’ ability to main-tain its position in favourable patches, suggest that a)individuals in habitat fragments will not show excessiverelatedness and consequently kin competition will notbe strong, and b) ‘accidental’ losses of individuals to

Fig. 7. Data used toparameterise individuallybased simulation models ofmovement for male andfemale P. argus : (a)proportion of time dedicatedto flying for each hourbetween 8:00 am and 8:00pm, (b) the distribution ofmove lengths (distancebetween stops), (c) thedistribution of displacementrates for individual movesand (d) the distribution ofturn angles between moves.

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Table 6. Observed and expected recaptures in target patch (alldays) predicted by null models. Model 1 assumes randommovement and complete permeability of internal and externalboundaries. Model 2 is the same as model 1 except that theinternal patch boundary was assumed to be impermeable.Asterisks show significance levels of chi-square tests with onedegree of freedom (observed and expected recapture frequen-cies outside target patch not shown). n.s. not significant, *P�0.005, ** P�0.001.

Model 1 Model 2

Population ExpectedSex Observed Expected

Local Male 6.2***23 5.4***Female 25 21.3 n.s. 25.7

Fragmented Male 18 9.0** 13.9 n.s.Female 43.710 36.8

8.9***Continuous Male 25 8.9***Female 17 28.5 30.2

of ants or some other type of cue. Not all myrme-cophilous Lycaenids oviposit in response to host ants(Pierce and Easteal 1986), and there is debate whethereven obligate mymecophiles such as the Maculinea spe-cies oviposit or select habitat in response to host ants(van Dyck et al. 2000, Thomas and Elmes 2001). InDonana, patches of L. niger are associated with rela-tively humid soil conditions (unpubl.), so P. argus mayuse other cues correlated with soil humidity to locateand recognise ant patches. We can rule out nectarsource density as a cue for habitat selection, becauseboth males and females moved out of the release squarewith higher nectar source density and no L. niger, butremained in the square with lower nectar source densityin the target patch. In Donana, P. argus shows a strongtendency to oviposit on host-plants associated with L.niger (Rodrıguez et al. 1991). Indeed, we have shownthat the presence of L. niger is by far the best predictorof the presence of eggs on host plants, and only weaklyinfluenced by the size or shape of the host plant (un-publ.). L. niger leaves strong scent trails when foraging(K. Fiedler, pers. comm.), so it is possible that P. argusis indeed using ants as cues for oviposition and habitatselection. A number of Lycaenids have been shownexperimentally to use ants as cues for oviposition, suchas Jalmenus e�agoras in Australia (Pierce and Elgar1985) and Hemiargus isola in North America (Wagnerand Kurina 1997).

Orientation to patches

Male P. argus from the local population released out-side the target patch were recaptured at significantlyhigher frequencies than expected from a random move-ment model, showing that patch search behaviour wasnon-random. It is not clear whether this non-randomsearch was homing behaviour particular to individualsreturning to their resident patch (navigation using fa-miliar cues), or if it was a general response to being insub-optimal habitat (systematic search, or navigationusing general cues of habitat quality). Both homingbehaviour and systematic search behaviour in responseto novel non-habitat have been observed in the meadowbrown butterfly Maniola jurtina (Conradt et al. 2000).Individuals from the local population were the first tobe released, so the null hypothesis may have been easierto reject for this group for two reasons: firstly, thisgroup had the most time available for recapture overthe course of the experiment, and secondly, the popula-tion density of resident P. argus in the target patchincreased over time, potentially reducing the targetpatch recapture rate of subsequent releases. Given thatour test for patch location by non-random movementwas very conservative (i.e. assumed no mortality and

the surrounding matrix will be low. As a result, onemay expect there to be little selection pressure forincreased emigration tendency from fragmentedpatches. In addition, the rate of movement of individu-als between different areas of the park may be too greatto allow local adaptation to occur. Our GIS model ofL. niger distribution in the scrubland area of Donanapredicts that patches of L. niger in the park may varyconsiderably from around a hectare or less up to hun-dreds of hectares in the lower elevation areas of thepark (unpubl.). However, patch isolation is relativelylow, with patches rarely being separated by more than200 m from other patches. Although such short dis-tances of non-breeding habitat may isolate patches ofP. argus in Britain (Lewis et al. 1997), the relativelylarge displacements we have observed in P. argus inDonana suggest that gene flow is probably high enoughbetween fragmented and non-fragmented areas toswamp any local selection due to fragmentation effects.In the more fragmented habitat, we have observeddisplacements up to around 900 m (fortuitous recaptureoutside study area), and in a less fragmented area wehave recorded displacements up to 783 m during acapture-mark-recapture study in a 1 km2 study area(unpubl.).

Habitat selection

Our results show that P. argus males and females areable to identify and maintain their position withinpatches of L. niger, as demonstrated by the significantassociation of recaptures of released individuals withthe target patch. Clearly this is an important behaviourfor an obligate Lasius myrmecophile such as P. argus(Jordano and Thomas 1992). However, it is not clearwhether P. argus is responding directly to the presence

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complete recapture of released individuals) and thatobserved recapture frequencies in the target patch wereactually greater than expected for females from thelocal population and males from the fragmented land-scape, it is probable that non-random search is a gen-eral response of P. argus to the absence of ants.

Patch boundary permeability

Plebejus argus males released into the target patchshowed a strong tendency to remain within the limits ofthe target patch, more so than would be expected by asimple diffusion process. Males may maintain theirposition in the target patch by constantly re-orientatingthemselves towards the conspicuous heathland vegeta-tion in the centre of the patch, or orientate to cuesproduced by virgin females emerging from L. nigernests, or the ants themselves. It is unlikely that nectarsource and conspecific density are important factorsinfluencing male movement, since males moved rapidlyaway from release site A where there was higher flowerabundance and large numbers of females. Even thoughother specialist butterflies such as Speyeria idalia havebeen shown to respond to low contrast patchboundaries (Ries and Debinski 2001), we show thatmale P. argus is capable of maintaining its positionwithin a patch whose boundaries are not delimited by aperceptible contrast in vegetation structure. Females areless active and less conspicuous than males, making theexpected target patch recapture frequencies predictedby the null model higher (less time to disperse) and theobserved recapture frequencies lower (harder to detect)than males. However, we may expect females to havefewer benefits from maintaining their position in L.niger patches than males. In L. niger patches, malesadopt a patrolling behaviour, presumably to maximisetheir encounter rate with virgin females emerging fromant nests. After mating, the eggs need several days todevelop before they are ready for oviposition, so fe-males have less urgency in maintaining their position inL. niger patches. Indeed, mated females may even incura cost maintaining their position in patches from maleharassment, resulting in relatively high emigration ratesfor females. Male harassment has been shown to influ-ence the movement behaviour of the butterflies Eu-phydryas anicia (Odendaal et al. 1989) and Proclossianaeunomia (Baguette et al. 1998).

Species whose distributions are limited by the re-stricted distribution of their mutualists may form animportant class of fragmented populations character-ised by low contrast patch edges. We have shown thatP. argus is able to respond to patches of mutualists withvery low contrast boundaries by increasing residencetime within patches (as shown by the strong associationof recaptures with the target patch and relatively shortdisplacements of butterflies released in the patch), and

for males at least, by using special behavioural mecha-nisms to locate and maintain their position withinpatches of mutualists. Such behavioural adaptations toliving in a naturally fragmented habitat may help P.argus to survive in patches isolated by anthropogenichabitat loss, and leads to the possibility that frag-mented populations of P. argus may be managed bymanipulating the distribution of host ants. Our studyalso suggests that extremely subtle changes in habitat,imperceptible to the human eye, may have importantconsequences in the migration rates of species. Thishighlights the need for obtaining a better picture of theprecise habitat requirements of the more endangeredspecies to understand their persistence in fragmentedlandscapes.

Acknowledgements – We thank the Parque Nacional de Do-nana and the Estacion Biologica de Donana (CSIC) for theirco-operation and support. In addition, we thank EnriqueDoblas, Pilar Fernandez and Paqui Garces for assistance inthe field. Itziar Lopez and Isabel del Rıo kindly identified thenectar plants of P. argus. Ilkka Hanski provided useful com-ments on an earlier version of the manuscript. The project wasfunded by a Training, Mobility and Research network grant(No. ERB FMRX CT98-0227) from the EU to Diego Jor-dano, and a grant from the former Spanish Ministry ofScience and Education to David Gutierrez.

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