At Home onForeign Meadows:

theReintroduction oftwo Maculinea ButterflySpecies

CENTRALE LANDBOUWCATALOGUS

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Prof. Dr. H.H.T.Prin s Hoogleraar in het Natuurbeheer ind eTrope ne n Ecologieva n Vertebraten Wageningen Universiteit

Prof.Dr . P.M.Brakefiel d Hoogleraar Evolutiebiologie Universiteit Leiden

Promotie commissie:

Prof. Dr. L.E.M.Ve t Wageningen Universiteit

Prof. Dr. J.M.va nGroenendae l Katholieke Universiteit Nijmegen

Dr.J.A . Thomas NERC Centre of Ecology and Hydrology, England

Dr. M.M. Kwak Rijksuniversiteit Groningen y; VX.JO27XV ,3o ->

yAfHom e onForeign Meadows: theReintroduction oftwo Maculinea ButterflySpecies

Irmgard Wynhoff

Proefschrift

ter verkrijging van degraa dva n doctor opgeza g van de rector magnificusva nWageninge n Universiteit Prof. Dr. Ir. L. Speelman, in hetopenbaa r te verdedigen opvrijda g 5oktobe r 2001 des namiddagst evie r uur ind eAul a Abstract

Wynhoff, I.,2001 . At Home on Foreign Meadows:th e Reintroduction oftw o MaculineaButterfl y species. Doctoral Thesis. ISBN90-5808-461-2 . Wageningen Agricultural University, The .

Maculinea live asobligat e parasites of specific Myrmica hostant s in meadow and heathland habitat maintained by low intensity landuse.Change s in agriculture causedth edeclin e and extinction of many populations. InTh e Netherlands, Maculinea nausithousan d M.te/e/t/s disappeare d inth e 1970s.I n 1990,the ywer e reintroduced following the recommendations ofth e IUCN.Thi s study focuses onth eevaluatio n ofthi s reintroduction intoth e nature reserve Moerputten inth e province of Northern Brabant. Population establishment and dispersalwer e monitoredan daccompanie d by researcho nth e impact ofth e reintroduction onspecies-specifi c genetic composition and behaviour.Maculinea teleiusimmediatel y established itself onon e meadow, where the population still occurs today. Maculineanausithous, beingth e more mobile species, colonized habitat patcheswit h thespecifi c host ant at higher distances.Thre e subpopulations werefounded . Atth e small spatial scaleth efemale s of both species were ablet o select habitat patches with host plants and host antst odeposi t their eggs, rather then loosing manyoffsprin g by random oviposition.A tth e ladscape scaleth e occurence ofth e populations was also strongly relatedt oth e presence ofth e specific host ant species. However, through the limited dispersal capacity ofth e adults highqualit y patches atgreate r distance remain uncolonized. Genetic analysis showedtha t the new populations have notexperience d a bottleneck in numbersfollowin g thetranslocation . However, the populations experienced selection duet ochange d ecologicalcondition s atth efounde r site, but ecologically relevant traits were apparently notaffected . The conservation ofth e reintroduced population should concentrate on keepinga high habitat quality onth ecolonize d sites andenablin g the development of metapopulations by improving the management of potential sites within colonization distance. Ultimately, the persistence ofth e populations depends on theavailabilit y ofa nextensiv e network ofsuitabl e habitat patches.Thi s requires conservation management at alandscap e scale.Thi s study emphasizes thata carefully planned and implemented reintroduction can bea successfu l toolfo r species conservation if bothsmal l and large spatial scales aretake n into account.

Doctoral Thesis (2001), Department of EnvironmetalSciences ,Tropica l Nature Conservation andVertebrat e Ecology Group,Wageninge n University, Bornsesteeg69 ,670 8 PDWageningen ,Th e Netherlands.

ISBN 90-5808-461-2 ^(GoS1^, 30%

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behorende bij het proefschrift: At home on foreign meadows the reintroduction of two Maculinea species

1 Poolse en Nederlandse Myrmica mieren spreken dezelfde taal (dit proefschrift).

2 Eencombinati e van een hoge dichtheid aan waardplanten en een hoge dichtheid aan waardmieren levert een slechte leefomgeving voor mierenblauwtjes op (dit proefschrift).

3 Metapopulaties van zeldzame en bedreigde soorten in het Nederlandse landschap hebben nietzozee r lastva n onvoorspelbare tegenslagen veroorzaakt door demografische, omgevings- en genetlsche factoren, maar veel meer van gebrek aan ondersteuning op beleids-e n uitvoeringsniveau (dit proefschrift).

4 Restoration ecology heeft in Nederland nogwe l eens negatieve gevolgen voor de natuur, omdat over het hoofd wordt gezien dat menwerk t aan relatief weinig verstoorde eilandjes inee n sterk verstoorde omgeving, en niet aan sterk verstoorde eilandjes in een weinig verstoorde omgeving.

5 Het kan niet anders of Tijmblauwtjes uit open, montane populaties zijn in staat om nun waardmieren te detecteren.

6 Degrootst e bijdrage aan Flamenco ind e afgelopen eeuw, wat de tekst betreft, is gedaan door Frederico Garcia Lorca (1898-1936).

7 Pecunia non oletse d non habet dulcem odorem aeternae laetae vitae (met dank aan Marian Picker).

8 Het is noodzakelijk om de anonimiteitva n zaaddonorschap te verbieden zodat kinderen het recht hebben nun vader te kennen.

9 Mobieltjes makend e mens minder mobiel.

10 Een afzuigkap is een belediging voor een goede kok.

11 Bei Niederlandern kommt der Wink mit demZaunpfah l nur dann an,wen n man den Adressaten mit dem Zaunpfahl erschlagt.

Irma Wynhoff Wageningen, oktober2001 Flugeltei n kleiner blauer Faltervo mWind e geweht, Ein perlmutterner Schauer, Glitzert,flimmert ,vergeht .

So mitAugenblicksblinken , So imVoruberweh n Sanic h das Gluck mirwinken , Glitzem,flimmern ,vergehn .

Hermann Hesse

furmeine Mutter Content

Chapter1: General introduction 9 Habitatfragmentation, environmental deterioration and thedecline of species 11 Declineand extinction ofMaculinea teleius and M.nausithous in theNetherlands 14 Ecologicalrestoration andreintroduction 16 Population geneticsof endangered species with smallpopulation size 18 Myrmecophily 20 Ecologyof Maculinea teleiusand M. nausithous 24 Habitatof Maculinea teleiusand M. nausithous 27 Ecologyof 30 Ecologyof Myrmica ants 32 Thestudy site 34 Aimsand outline of this thesis 39

Chapter2: Therecent distribution ofthe European Maculinea species 43

Chapter3: Population development of the Butterfly species Maculineateleius and M. nausithous afterreintroduction 59

Chapter4: Oviposition byMaculinea nausithous andMaculinea teleius butterfliesin relation to their host plant and specific host ants 75

Chapter5: Looking for the ants: Habitat selection oftwo Maculinea butterfliesand their Myrmica hostants 95

Chapter6: Landscape constraints in thedispersal of two parasitoid butterfly species 117

Chapter7: Genetic variation isretained after reintroduction ofthe butterflies

Maculinea nausithous andMaculinea teleius 135

Chapter8: Synthesis 157

References 189 Summary 203 Samenvatting 209 Zusammenfassung 220 Thankyou, dankje wel, Danke,gracias! 229 Curriculum vitae 233 Generalintroduction 10 Generalintroduction

Nature pays a high prizefo r the increase innumber s of human beingsal l overth eworld .At leasta thousan d plantan danima l species havebee n drivent o extinction by peoplejus t since 1600A D (Jenkins 1992),an dthi s is probably only a parto fwha t is lostforever . Many morespecie s still survive butar efacin g the samefate ,soon .Wh y do so many species go extinct? Mostwer edweller so fsmal l islands andwer e simply harvested untiln o individuals were left. Other small island species were unable towithstan d introduced predators.Th e mainland situation isdifferent . Here many species arethreatene d with extinction because,apparently ,the y haveto o narrowa tolerancefo r environmental change aswel la s needing aver y specific combination of ecological conditions.Yet ,fe w ofthes e species are extinct already, andwit h enduring efforts these species may still bepreserved . Maculineabutterflie s aretypica l for the latter group of species:the y have highly specific ecological requirements combinedwit h littletoleranc e for change. Maculineabutterflies , ofwhic hfive specie s occur in Europe,sho wa typical adaptation ofthei r biology: during apar t ofthei r life cyclethe y parasitise the nestso f Myrmica ants (Thomas 1984).Thi s study concentrates onth eecolog y andconservatio n oftw ospecies , Maculinea teleius, the butterfly, and Maculinea nausithous, thedusk y large blue. Both speciesfrequentl y live sympatrically onth e same meadows.The y use thesam e hostplant, Sanguisorba officinalis, and during their parasitic life stagesthe y behave ina simila rwa y inth ean t nests (Thomas 1984,Thoma s &Elmes1998).

Habitatfragmentation, environmentaldeterioration andthe decline of species

Although there are indications that startedt odecreas e in numbers and distribution early inth etwentiet h century,the y have declined throughout Europe most markedly during the pastfe w decades. Many regional and national reviews aswel l ascas e studies are presented in Pavlicek-van Beek etal. (1992) and Pullin (1995).Accordin gt o national Red Data Lists,abou t40-50 %o f all Lepidoptera species occurring in andAustri a areendangere d and2-5 %o fth e species occurring inthes e countries have become nationally extinct (Erhardt 1995). InTh e Netherlands thesituatio n iseve nworse :24 %o f the sedentary species have gone extinct and43 %ar ethreatene d (Wynhoff &va n Swaay 1995). Especially inth e

11 Trend J3L deer-75-100 % J3U deer.50-75 % deer. 25-50% deer. 15-25% O stable 9 unknown

Figuur 1.1:Abundanc ean dstatu so fMaculinea teleius inEurop e(afte rVa nSwaa y & Warren 1999). Abundancei sgive ni npercentag eo foccupatio no fcountr ysurface ,i n whitecountrie sth especie si smissing .Tren di sgive ni ndevelopmen tdurin gth elas t2 5 years.

Central andWes t European lowlands many butterfly habitats are endangered andspecie s arethreatened .Onl y inth eAlpin e and Mediterranean region doesthi s decline seemt o be less severe (Erhardt 1995, Munguira 1995). Thefiv e Macu/Znea-species(M. arion,M. teleius, M. nausithous,M. alcon and M.rebel!) represen t asmal l group of highly specialised and sensitive butterflies that are currently being severely endangered. Inth e majority of countrieswithi n their rangeth e abundance is lesstha n 15%an dwithi n countries often even lesstha n 5%o f the area.Th e comparison between the abundance 25year sag o andth e present abundance shows adecreas eo f

12 Abundance <1% -M 1-5% m 5-15% m >15% Trend deer. 75-100% XL deer. 50-75% deer. 25-50% deer. 15-25% o stable 9 unknown

Figuur 1.2:Abundanc e and status of Maculinea nausithous in Europe (after Van Swaay &Warre n 1999).Abundanc e is given in percentage of occupation of country surface, inwhit e countries the species is missing.Tren d is given in development during the last 25years .

20t o 50%fo rfou r ofthes e species,wit h Maculinea arionexperiencin g a decrease of 50t o 80%i n Europe (Van Swaay &Warre n 1999).Th e present trend and status of Maculinea teleiusi sgive n infigur e 1.1,fo rMaculinea nausithousse efigur e 1.2. Maculinea arion,M. teleius an d M.nausithous are already mentioned inth eAppendi x IIo fth e Bern Convention,an d iti s recommended to include also M.alcon an d M.rebel! (Va n Swaay &Warre n 1999). Iti sobviou s that conservation measurements must betake nt o ensure longter m survivalo fthes e species (Munguira& Martin 1999).

13 Declineand extinction ofMaculinea teleius andM. nausithous inthe Netherlands

Both Maculinea species have most likely hada restricteddistributio n in Western and Central Europe since scientific research began,bu t locally they could bever y abundant. Thewe t meadows inth e valleys of the riversystems, which represented their habitat, used tofor m acontinuou s landscape where refuges were always present enabling survival of catastrophic events sucha s inundations or mowingdurin gth efligh t period.Mos t likelyth e primary habitat inth e beginning ofth e Holocene also consisted of long bands of rough vegetation inth egradien t between riverine vegetation andfores tedge swit h locally wet anddr y patches and quitedynami c characteristics. Metapopulations must also have existed insystem s ofope n patches in forests that dominated the landscape some 10,000year s ago (Prins 1997). Extended open grasslands were almost absent, thereforeMaculinea butterflies must havebee nquit e localised. Iti s likelytha tthe y existed in dynamic metapopulations onconstantl y changing networks of patches. Probably the increase in human population density andth e development of slash and burn agriculture around 5,000 B.P. resulted in more patches with openvegetatio n to becolonise d byth e butterflies. Inth e MiddleAge s (700 B.P.) manyfores t areas were cleared and usedfo rfield s and meadows. Furthermore, haymaking became increasingly common (Prins 1997). These early improvements inagricultura l activities created anope n but patchy landscape with many suitable placesfo r various butterfly species. Amongst others, Maculinea nausithous and M.teleius mus t have increased in numbers of populations and in numbers of butterflies per population.Maculinea teleius also occurred at borders of peat bogs,whic hwer e to a lesser extent influenced by humanactivities . However,wit hth e later "agricultural improvements", that started inth e 1960s,th e beneficial effect of low intensity farming camet oa n end (Ebert& Rennwald 1991). Intensification of management byfertilisatio n and increasing ofth e mowingfrequenc y resulted inth e loss of many plant species andlevellin g ofth e vegetation structure.Cessatio n of traditional agriculture andabandonin g of meadows favours a roughvegetation ,whic h in many cases is notfavourabl e for butterflies. Meadowswer edestroye d by ploughing andgrowin g crops.Other swer e mownfrequentl y during thefligh t season which resulted inremova l of eggsan d caterpillars.Th e surviving females were notabl et o deposit eggs anymore because hostplants were missing.Anothe r reasonwa sth e increase ofgrazin g intensity which hada

14 disastrous effect onth e Myrmica host ant populations. Maculineapopulation s were also lostthroug h housebuildin g and new infrastructure. Innatur e reserves, populations were destroyed by mowingth ewhol e area inth e beginning ofAugus t because mowing attha t time ofth eyea r is considered most beneficial for the conservation and restoration of a species-rich vegetation.Thu s butterflies even became thevicti m of nature restoration projects.Anyhow , this causedth edisappearanc e of both Maculinea species fromth e nature reserve Moerputten inth e centre ofTh e Netherlands,wher e they were later reintroduced. The last indigenous Dutch populations of Maculinea teleiusan dM. nausithoususe dt othriv e ina nare a called "Elfenmeertje" closet oth e small village of Herkenbosch inCentra l Limburg. Eventhoug h Lepidopterists had warned nature conservation organisations, this areawa s not bought and preserved. Inth e beginning ofth e 1970s,th e development of a camping place started,whic h resulted inth e extinction ofth e last populations (Geraedts 1986,Ta x 1989). Maculinea teleiususe dt ooccu r inth ecentra l partso fth e province of

c^

Figure 1.3:Distributio n of Maculinea teleius and Maculinea nausithous inth e Netherlands from 1870t o 1976 (from: Database Dutch Butterfly Conservation). Circle: Maculinea teleius, dot: Maculinea nausithous

15 Limburgaroun dth e cities of Roermondan dVenl o (figure 1.3). Inthi s area thespecie s wasfoun d inth e riverine grasslands alongth e River Maas (Meuse)an dth e streams Roer, Swalman d Schandelosche Beek.Th e populations inth e province of Northern Brabant were located inth e stream valleys around Oisterwijk and inth e region between Oisterwijk and Breda andValkenswaard . Thefe w populations inOverijsse l were isolated from these (Tax 1989). The distribution of Maculinea nausithouswa st o a large extent the same asth e one of Maculinea teleius(figur e 1.3).However , there were no populations inth e northexcep tfo r theon e inth e Hasseler Beek close toth e city of Hengelo (Tax 1989).

Ecologicalrestoration andreintroduction

InWes t Europe, most modern day Maculineahabitat s are of secondary nature,suc ha sdifferen t types of meadows or heathland in agricultural landscapes.Suc h habitat patcheseasil y loosethei r qualityfo r butterflies if the habitat management changes. Under such changes, effects onth e delicate interplay of Maculinea butterflies, Myrmica antsan dth e vegetation aret o beexpected .Som e kindo f (agricultural) activity istherefor e needed to keepth e habitats suitable for the species. Inth e management of nature reserves,th esam eaim sa sachieve d bytraditiona l land useshoul d beaime d for. However, there are often conflicting management scenario's for different species sharing the samelocation . Butterfly populations have often disappeared due to changes in management of sites, as has happened withth e Maculineas inth e Moerputten nature reserve. Itseem s very attractive toowner s andwarden s of nature reserves to restore the habitat sites and reintroduce the lost species again,particularl y ifth e concerned species are attractivet o the general public. Reintroduction ortranslocatio n of species is by no means new. It has beendon efo r many species,sinc e manyyear s ago and almost all over theworld . InAmerica ,amongs t others,th e grey wolf has been reintroduced ina number of States (Bangs era/. 1998, Noss etal. 1999),th ewol f was brought back toth eYellowston e National Park (Frittse t al. 1997)an dblac k bearswer e released inArkansa s (Clark &Smit h 1994, Smith &Clar k 1994). Large herbivores were reintroduced (Bison and elk: Truett 1996,caribou : Pitt &Jorda n 1994))a swel l as small species (otter: Swimley etal. 1999) . In Africa, many large and medium-sized mammal species have been

16 reintroduced into nature reserve after their local extinction duet o overhunting. Inman y of these cases, resource competition between the introduced species and a resident species of approximately the same body weight may have played a rolewhethe r a reintroduction succeeded orfaile d (Prins &Olf f 1998).Severa lfis h and bird species have also been subject toa reintroduction project (Byrd era/. 1997,Foi n etal. 1997,Winnemiller & Anderson 1997). In Europe,afte r severe habitat destruction,th e beaver has been reintroduced in many countries,fo r instance inSwede n (Ellegren efal. 1993),Th e Netherlands (Nolet & Baveco 1996),Scotlan d (Macdonald etal. 2000, South etal. 2000),German y (Rieder & Rohrer 1982), (Stacker 1984, 1985),Czechi a (Kostkan etal. 1997) and (Grubesic & Mayer 1996). Much attention has beengive nt o reintroduction ofth e lynxa s top predator ina number of regions (Breitenmoser & Haller 1993, Scott efal. 1999,Stah l& Vande l 1999). Inth e Netherlands, bringing backth e lynx toth e nature reserve "HogeVeluwe "appeare d to be notfeasibl e (DeJon g etal. 1997).Th eai mo f these reintroductions isgenerall y the restoration of ecological processes.A t least within the published papers,thi s seems to be possible after goodfeasibilit y studies have been conducted and no potential problemsfo r thefounde r populations could bediscovered . However, even when applied according toth e recommendations ofth e IUCN,succes s isno t guaranteed. Publications mainly evaluate successful reintroductions, while only afe w failures of reintroductions ortranslocation s were published. Inbutterflies , reintroduction seems to bequit e difficult. Oates &Warre n (1990) showed,tha t newlyfounde d populations mostly become extinct within five years after release.Th efailur e isofte n due toecologica l requirements of thespecie s which are not met inth e reintroduction area,o r by translocating only aver y low number offounder s (e.g.Dempste r & Hall 1980).Th e number of successful (re)introductions istherefor e limited. Afamou s example for asuccessfu l reintroduction of a butterfly species is the case of Maculinea arioni n England.Ver y large populations ofth e ant species Myrmica sabuletiar e neededt osuppor tth e parasitic butterfly whose caterpillars feed onan t grubs and hibernate inthes e ants' nests.Adequat e densities ofth e host ant species only occur onwar m siteswit h aver y short sward kept by high levels of domestic grazing.Throug h cessation ofgrazin g and decline of rabbit densities thevegetatio n sward grew up. Myrmica sabuletidecrease d innes tdensitie s and Maculinea arionwen t extinct. Following successful research intoth e complicated relationship between butterfly and hostan t andth e impact of management, nature reserves were restored. Underth eol d grazing level,th e ant population were ablet o recover

17 andth e butterfly wassuccessfull y reintroduced (Thomas 1989, 1991, 1995, Thomas & Elmes 1992). In 1989th e Ministry ofAgricultur e and Fisheries decided to reintroduce both Maculinea species inth e Netherlands.A feasibilit y study showedtha t in the Moerputten nature reserve the only host plant Sanguisorba officinaliswa s very abundant and both host antspecie swer e present. Itwa s possible to prevent mowingo foccupie d meadows betweenth e beginningo fJun e and the middle of September. Then the lost species were brought backt o their former sites.O nth ewar m evening of July 30th, 1990, 33 malesan d5 3 females ofM. teleius,an d 22 males and4 8female s ofM. nausithous, capturedfro m a metapopulationi n ,wer e released onth e meadows of the Moerputten (Wynhoff 1991, 1992,Wynhof f &Va n der Made 1995). Populations of both species are now presentagain . Forthes efounders ,th e reintroduction iscomparabl e to a population bottleneck:the y are physically isolated from any other metapopulation and therewil l ben oexchang eo f individuals from anysource .Suc h isolated populations have a higher chance of stochastic extinction compared to populations embedded ina metapopulatio n system. Duet oth e geographically isolated position ofth e reintroduction site,th e introduced populations havet o survive without any possibility of exchange of individuals and geneflow .Afte r havingsettled ,number s havet o increase inth e nature reserve.The n by means of local dispersal,othe r ecologically suitable sites havet o becolonised .Th e aimo f any reintroduction of species withina natural system must bet o establish a metapopulation system consisting of severalsubpopulation s ofeac ho fth e Maculinea species ora ver y bigsingl e core-population. Only thenther e isa chanc e of long-term persistence.

Population geneticsof endangered species with small population size

The reintroduction of a population to anempt y habitat patch leadst o conditions underwhic h the exchange of individuals between sites is reduced or impossible. Consequently, gene poolsar ethe n separated and,unde r pressure ofdifference s inselection ,ma ystar tt oevolv e indifferen t directions. Inan y population, individuals with a highfitnes s atth e new site contribute more progeny toth e next generation compared to individuals with lowfitness . The processes leading to differences in reproductive success of individuals within a population involve random effects (sucha so fweather ) and natural selection,whic h is brought about by components ofth e environment. Evolution occurswhe nther e isa chang e inth e characteristics of a population

18 and ingen efrequencie s over a number of generations duet oselectio n or random effects (Hartl& Clarke 1989, Brakefield &Shreev e 1992). Furthermore,founde r populations involved in reintroductions are usually low in numbers of individuals. Populations being reducedt o aver y small size experience losso f genetic variation through loss ofvariant s ofgenes ,o r alleles (Gilpin 1991, MaynardSmith1998) . Such events, involving chance rather than selection,ar edescribe d as genetic drift. Inth e meta-population situation such losses may be compensated for by immigration of individuals fromothe r subpopulations. Reintroduction creates asituatio n inwhic h losses through chancear e expected for the progeny ofth efounders :ther e will most likely bea decrease of genetic variation and loss of alleles compared toth eorigina l source population (Hartl &Clark e 1989, Maynard Smith 1998).Ther e isn o counterbalance through immigration and gains through mutation happens o rarely that they can beneglected .Th eamoun t of loss isals o relatedt oth e duration ofth e bottleneck. There will befewe r problems,whe nth efounde r population can rapidly increase insiz eafte r having passed a bottleneck (Saccheri efal. 1999) . Another effect ofth e decrease in numbers of reproductive individuals by means of reintroduction could betha t there isa highe r chance of inbreeding (Hartl & Clarke 1989, Maynard Smith 1998).Whe n sibs are not ablet o recognise eachothe r and mate randomly,the nth e chance of sib-mating decreases with an increase in population size.Withi n any butterfly population notw o individuals are identical, but sibs resemble each other moretha ntw o randomly chosen individuals. Therefore inbreeding increases homozygosity, andthereb y may lower thefitnes s of individuals (Saccheri efal. 1998) .Th e losso ffitnes s at individual level may leadt o an increased risko fextinctio n of the population concerned.Thi s effect isundesirabl e ina reintroduced population.Change s ingeneti c variation can bestudie d atth egenotypi c or phenotypic levels,o r onan y seto f individual characteristics resultingfro m interactions between genes andth e environment during development (Brakefield &Shreev e 1992). Even ina specie s sucha s Maculinea nausithous, which iscomparabl y constant in its phenotype and has not evolved anysubspecies ,difference s inwin g size,spo t number and spot size between individuals and between populations are easily detectable. Furthermore,studyin gth e phenotype isofte n lessdestructiv e to a population compared to studying thegenotype .

19 Myrmecophily

Butterflies ofth efamil y engage ina rich andfascinatin g diversity of interactions with other species.Althoug h as adultsthe y behave astypica l butterflies, as larvae many lycaenids inhabit asequenc e ofver y different niches.Severa l species are carnivorous as larvae;som efee do n homopterans,whil e relationships between othersfor m essentially parasitic relationswit h ants, living inthei r nestsan dfeedin g onth e antgrubs . Lycaenids and ants spanth e continuum of positive and negative interactions: from predation or parasitism (plus/minus) through commensalism (plus/zero) totru e mutualism (plus/plus) (Bristow 1984). The evolutionary success of myrmecophily is based onth e appeasement ofth e aggressiveness of antsan dth e possibility to use itfo r ownprotection . Butterfly specieswit h a myrmecophilic life style maygai n selective advantages over non-myrmecophilicspecie s (Fiedler & Maschwitz 1988) which can beo fgrea t importance inecosystem s with high species numbers and nestdensitie s of ants, sucha s tropical rainforests . Incolde r climates, myrmecophily is lessfrequen t (Fiedler 1991).

Myrmecophilous organs Duringth e evolution of myrmecophily, several specific larvalorgan s have developed.Thre e types of larval organs play major roles inth eant-lycaeni d interactions: • pore cupola organs (=lenticles) They are scattered overth ewhol e body surface. The (volatile?) secretions ofth e pore cupola organs are supposed to appease theant s that encounter lycaenid caterpillars or pupae (Malicky 1969, Pierce 1983, 1984, Cottrell 1984). • dorsal nectary organ (=Newcomer's gland,=hone y gland) The dorsal nectary organ is located onth e seventh abdominal segment. It produces watery secretions containing mainly dissolved carbo-hydrates in concentrations of about 15% (Maschwitz etal. 1975 , Pierce 1983, 1984). Ina mutualisti c symbiosis withth e ants protecting the larvae against predators or parasitoids,th e ants may gain important nutritional benefits from thesugar y secretion (Fiedler & Maschwitz 1988). Furthermore, it induces recruitment of nestmatesan destablishe s more or less permanent trophobiontic associations with the myrmecophilous lycaenid larvae. • retractiletentacula r organs (=retractile tubercles, =lateral organs) Thetentacula r organs are a pair of extrudable epidermal tubes located on the eighth abdominal segment, covered with numerous branched hairs.

20 Thetentacula r organsoccu r in myrmecophilous lycaenid larvae,whic h also possess adorsa l nectary organ but insom e species either dorsal nectary organo rtentacula r organs are missing (Kitching & Luke 1985, DeVries etal. 1986) .The y are extruded and retracted repeatedly when the attending ants palpate alarv a intensively (Malicky 1969).Th e species-specific reaction and behaviour ofth e ant species concerned is caused bya chemica l stimulus, possibly analar m pheromoneo ra n analogous substance (Henning 1983, Kitching & Luke 1985).Th efunctio n is probably allomonal. Incertai n taxa ofth e Polyommatinae,th e tentacular organs arethough t to produce volatile compounds that appeart o mimic someo f the alarm pheromone signals ofth e ants (Cottrell 1984).

Types ofmyrmecophily Within the European Lycaenidae, many types of relationships with ants occur. Myrmecoxenousspecies , such as most ofth e genus Lycaena, do not show any relationship with ants at all, butthank s tothei r pore cupola organs they areals o notattacked . Fromth eweakl y myrmecophilous species (hairstreaks), very few antassociation s have been reported and stable ant-associations areforme d only exceptionally. The larvae ofth ecommo n species,fo r example Celastrina argiolus, have often been observed attended byants ,bu tthe y may also live solitary. They are classified as being moderately myrmecophilous. All Plebejus and Polyommatus species are steadily myrmecophilous: most, if not all, mature larvae live in association with ants. Many Lycaenids belongt othi s type (Fiedler 1991, Fiedler& Maschwitz 1989).Th e mostextrem e kindo f myrmecophily isfoun d inth e obligately myrmecophilous butterflies. Their larvae arecompletel y dependent on ants.Th e Cigaritis species live as commensales inth e ant nests (Rojo de la Paz 1992),specie s of Maculinea actually completethei r lifecyclea s parasites. The relationship between ants and butterflies is basedo nth e caterpillar's provision offoo d secretion withth e abovementionedspecialise d glands to ants inexchang efo r protection against enemies and parasites (DeVries etal. 1993). Inaddition ,th e caterpillars may also possess other specialised behavioural and morphological features that play arol e in maintaining symbiotic associationswit h ants,an d it islikel y thatthes e characters haveal l been selected for directly byth e ants.Ant s live in high numbers together in organised colonies and have highly developed communication systems. Thus,th e main problem forth e caterpillars ist o integrate intoth e communication systemtha t isspecifi c toth e ants.Th e close relationship with the ants,afte r having entered the nest,give s riset oth e idea that nextt o

21 other adaptations,som e kind of communication takes place.A s much ofth e social behaviour ofant s is released andcontrolle d by pheromones, chemical communication is likely totak e place (Henning 1983).

Adaptationsto myrmecophily in Maculinea InMacu//'nea-species ,th e relationship with ants isver y intense. Mosto f the lifetime of an individual isspen t inth e ant nests,bein g dependent onfoo d and/or caredelivere d byth e host ants.Thu s inth e morphology and behaviour ofth e caterpillars,difference s to non-myrmecophilousan d non­ parasitic species can befound . The caterpillars of all lycaenids have athic k andtoug h skint o protect them against ant bites (Malicky, 1969).Th e body shape isdifferen t fromth e general shape of caterpillars:vita lorgan s are protected behind protruding ridges containing non-vital organs (Cottrell 1984).Whe nfeedin g onan t grubs the Mact///nea-caterpillars pullth efron t ridgesove rth e head,thereb y hiding their prey.Worke r antsdon' t even seemt o noticetha t the caterpillars are feeding. Myrmecophilous caterpillars havedifferen t types of glands which produce various kinds of secretions to interest and attract antsan d atth esam etim e suppress low levels ofaggressivenes s (Cottrell 1984,Akin o era/. 1999).Thi s is probably the most important means bywhic hth e caterpillars deceive their hosts. Secretion may also be important as afoo d source forth eants .Al l Maculinea species possessonl y porecupol a organsan da dorsa l nectary organwhil e tentacular organs are missing.Kitchin g & Luke (1985) describe the myrmecophilous organs of Maculinea anon. Caterpillars having a myrmecophilous life style are very slow.The y also lackth e beat-reflex. Hence any increase inaggressivenes s inth eant s by rapid movements isavoide d (Cottrell 1984).Th e ultimate larval instar (L4)o f several Maculinea species imitate the behaviour of ant grubs to betake nt o the ant nests. Furthermore, caterpillars and pupae of Maculinea make species-specific sounds, resembling the sounds ofth e hostant s (DeVrieset al.1993 , Elfferich 1998).

Specificityoflycaenid -ant relationship Within Europe most lycaenid larvae are unspecific inthei r relationship withan ttax a andfacultativ e inth e sensetha t the larvae candevelo p welli n theabsenc e ofants .Thei r relationships show little specificity withinth e limits ofth e anttax a (Malicky 1969, 1970).Th e associations formed byant swit h most lycaenid species arecontrolle dfa r more byth eexten t towhic h the

22 habitat preferences ofth e lycaenidsoverla pth eforagin gterritorie s ofth e antstha n byan y specificity inherent toth e partners. Insuc h relationships the caterpillars benefitfro m protection from parasites and predators byth eants . They also experience lower levels ofan taggressio n andescap epredation , as antsar e important predators.Wit hsuc h aims,i t isbette r to beabl et o appease as many ant species as possible instead of beingdependen t on only afe w numberso fan tspecie s (Fiedler 1991).Th e Maculineaspecie s contrastwit hthi s inthei r specificity toonl y afe w species within thegenu s Myrmica.B yals o being restricted toonl y one hostplant or afe w hostplant specieswithi n asingl egenus ,thei r niches becomeeve n smaller. There must bea n evolutionary advantagefo r Macu//nea-species,t o simultaneously bedependen t ononl y afe w hostplant species and afe w ant species.Th e longesttim e spandurin gth e lifecycl e isth elarva lstage . Advantages of acomparativel y enemy-free spacedurin g the larval instars are evident. It is unknown, however, whythe y remaino nth e hostplant until the L4insta r andd o notleav e itearlier . Advantages could berelate dt oth e winterperiod.Change s in microclimate,especiall y during thewinter , are buffered inth ean t nest. However, there isstil la high mortality of caterpillars inth e ant nests (Thomas& Elmes 1998).The y canstarv e as a consequence of overexploitation or can bedetecte d as parasites andfe d upon byth eants . It isa questio n whether the advantages compensate forth e riskso fan t predation or starvation after thefoo d source available inth e ant nesti s exhausted. The intense specialisation of Maculinea butterflies andthei r association with specific Myrmicaspp. mean s anextr a riskt o any reintroduction project. For Maculinea alconth e host specificity differs between regions (Elmes efa/ . 1997)bu tfo r Maculinea teleius and M.nausithous w e have no knowledge of suchspatia lvariation . Ifhos tspecificit y hasevolve dt o extreme specialisation, itcoul d meantha t the caterpillars from thefounder s are not ablet o break the communication code ofth e host ants atth e reintroduction site.Th esucces s ofth e reintroduction project couldthe n be influenced by the relatedness betweenth efounde r population andth evanishe d population atth e reintroduction site.

23 Ecologyof Maculinea teleiusand M. Maculinea teleius nausithous deposits the eggs on very young green Lifecycle of Maculinea teleiusand M. inflorescence of Sanguisorba officinalis. nausithous Eachflowerhea d is Maculineabutterflie s typically suitable for oviposition live inclose d populations with little for no more than 5t o 7 or nodispersal .Al l species are days. The females univoltine,wit hth e adultsflyin g for normally lay only one egg per flowerhead 2-4weeks ,dependin go nth e (Thomas 1984). geographical location andth esiz e ofth e population. Maculinea teleius and Maculinea nausithous areo n Maculinea nausithous uses the flowerheads of thewin g from mid-June to thefirs t jrf& Sanguisorba officinalis Jtj&§ week of September. Populations when theyjus t have ^(H yJP are knownt o have sizes from less begun to develop red mew than 50t o moretha n 5000 coloration. Large •py r individuals inon e summer. Onsite s flowerheads are ( with both species, Maculinea teleius preferred upon smaller ,/ ones. Usually two to isalmos t always the rareone . three eggs at atim e are being laid. The egg load can increase upt o 33, but only few caterpillars The eggs are deposited onth e will survive (Thomas 1984). only host plant Sanguisorba officinalis(figur e 1.4) (Thomas 1984). Howeverther e are marked differences inth e phenological statewhe nth eflowerhead s are used as an oviposition site byeithe r species or as anecta r source (Figurny &Woyciechowsk i 1998). The eggs are hidden between the budso fth e host plant.The y lack athic k eggshell to protect themselves against parasitoids (Munguira 1986).Almos t allegg swil l hatch intotin y caterpillars. Duringthei rfirs t instarsthe ywil l stay onth eflowerhead s andfee d as phytophagous onth e developing seeds.Ther e is nodifferenc e inth e behaviour between thetw o species.Th e caterpillar hollows outon e or moreseeds .Th e last moulting takes place in thetunne l thuscreated .O nopenin g aflowerhea d thetrace s of asuccessfu l survival untilth e early L4larva l instar will befound .Th etunne l inth e seed lastconsume d isfille d withth e skin ofth e penultimate L3-caterpillar, witho n onesid eth e head shield and onth e other sideth efaeces . The overall survival onth eflowerhea d from theeg gt oth e L4-caterpillar isfo rMaculinea teleius50 %an dfo r Maculinea nausithous 47%(Wynhoff , unpublisheddata) . From midJul yt o midSeptembe r the caterpillars liveo nthei r host plant.

24 The behaviour of the caterpillars For Maculinea teleius the main host ant species of Maculinea teleiusi sver y similar is , with in addition totha t of Maculinea arion(Thoma s Myrmica ruginodis in (Lhonore, pers. & Elmes 1998).O nth e flowerhead comm.), in Poland (Figurny, pers. of Sanguisorba officinalis, normally comm.)an d inCentra l Germany (Ebert & Rennwald 1991) and occasionally Myrmica only one egg isdeposited .Thi s vandeli and Myrmica sabuleti (SBN 1987, reduces food competition inth e Thomas era/. 1989). first tothir d larval instar.

Occasionally, upt othre e Maculinea nausithous is probably most caterpillars can befoun d onon e specialised to one host ant species. Nearly all flowerhead. However, later onegg s over Europe,th e host ant is Myrmica rubra L. of Maculinea nausithous can also (Bink 1992, Ebert & Rennwald 1991, SBN 1987, bedeposite d onth e same Thomas etal. 1989). Recently on a Polish site, survival in nests of Myrmica scabrinodis has flowerhead.A s aconsequence , been observed (Figurny, pers. comm.). Also one food competition between larvaeo f of the Spanish populations isa parasite of two species can occur. The Myrmica scabrinodis. caterpillars of Maculinea nausithous almost alwaysexperienc e foodcompetitio n asa consequenc e of the high egg load.The y reachth e L4-instar intw ot othre e weeks,whil eMaculinea teleiuscaterpillar s needthre e tofou r weeks.Th e caterpillars of Maculinea teleiusar e also biggerwhe nthe y leaveth eflowerhead . Having moulted toth efourt h instar,th e caterpillars drop off their hostplants ontoth e ground.The y havet o befoun d byworker s ofMyrmica- species, "adopted"an d takent oth e ant nests.Th efirs t contact between caterpillar and antdetermine s largely whether it hasa chanc e tofinall y develop intoa butterfly. The adoption procedure involves specific rituals and isdifferen t depending on Maculinea and Myrmicaspecies . Inth ecas eo f Maculineateleius i tca n lastfo r 30-90 minutes or even longer (Thomas 1984, Fiedler 1990).Th e caterpillar is palpated and in response offers droplets of secretionfro mth e dorsal nectary organ. Finally, after showing"humping " behaviour, it iscarrie d toth e ant nest.Th e adoption ritual of Maculinea nausithousi s much shorter. One important aspect of parasitism istha t only certain, species-specific Myrmicaant s can act as host. Both Maculinea species canonl y survive the winter whenthe y are adopted byth e correct antspecies . Maculinea teleius survives best inth e nestso f Myrmica scabrinodis, but Maculinea nausithous prefers Myrmicarubra (Thoma s etal. 1989) .

25 Once inth e ant nest, the caterpillars are ignored completely. Fromno w onvegetaria n food isno taccepte d anymore:the y change their diett o feeding onan tgrubs . Evenwhil e feeding onth e ant grubs,th eworke r ants do not react. In rearing experiments, the caterpillars do notsta y among the brood but prefert o beapart . Only occasionally havethe y been observed beingfe dwit h ant regurgitations. They receive only little attention andwil l not bedefende d or carried away in caseo fdisturbances .Therefore , both species areclassifie d as primitive parasites (Thomas &Wardla w 1992).Th e number of caterpillars of Maculinea teleiuspe r ant nest isalway s low, rarely exceeding one individual (Ebert & Rennwald 1991, Thomas etal. 1998) . Furthermore they areabl et o starve for a period.Thi s may bever y important becauseth e nests ofMyrmica scabrinodisar equit esmall .Th e larvae are ablet odecreas eth e number of ant grubs so heavily that the colony moves away.Th e caterpillars areabl et owai t for recolonisation ofth e empty colony site andthe ndeplet eth e next ant nest. Maculinea nausithous isabl et o survivewit h several caterpillars inon e host ant nest;thes e ants'colonies , however, aresevera l times bigger thanthos e of Myrmica scabrinodis (Wardlaw &Elme s 1996,Thoma s & Elmes 1987, 1998). The caterpillar will stay inth e nestfo r approximately ten months,feedin g from the antgrub s and hibernating. Moretha n 90%o f itsfina l body massi s gained inth ean t colony. Finally pupation takes place inth e upper chambers of the ants'nests .

oviposition on Sanguisorba officinalis

Maculinea nausithous egg Maculinea teleius larvalinstar s L,-L3

pupation Myrmica rubra Mynnicascabrinodis

Larval instar L4 and hibernation

Figure 1.4:Th e life cycle of Maculinea nausithous and M. teleius.

26 The Maculinea caterpillars canthemselve s be parasitised by Ichneumon and Neotypushymenoptera nwasps . These species arever y rare andwit h theexceptio n of Ichneumon eumerus parasitising on Maculinea rebel!, their ecology is notye t unravelled (Hochberg etal. 1996 , Hochberg efa/ . 1998, Thomas& Elmes 1993).

Habitat of Maculinea teleius and M. nausithous

Atfirs t sight, both Maculinea species seemt o share closely similar habitat types. However, after aclose r look,ther e are differences explaining the coexistence on many sites.Th e habitat of both Maculinea species can best bedescribe d as marshy lowland.Site s are often situated instrea m valleys or besides lakes. Most populations thrive onvegetations ,whic h canb e characterised as Moliniono r humidArrhenaterion (Eber t & Rennwald 1991, Rozier 1999,Wynhof f 1992). Inthes e humid meadowsth evegetatio n is mownonc e totwic e ayear . Maculineateleius i s more restricted to open vegetation,whil eMaculinea nausithous can also befoun d on roughvegetation ,suc ha s Maculinea teleius occurs in open Junco- meadowswhic h have been Molinion vegetation's inth e North via humid abandoned for several years Arrhenaterion to closed Phragmites or Carex vegetations inth e South of itsdistributio n range. (Bergmann 1952,Bin k 1992, Ebert Itcanno t be found in low quality habitat types. 1991, GeiRler 1990, Richard 1997, Sonnenburg 1996,Thoma s 1984, Wynhoff 1992, 1996). Ingeneral ,meadow s with low- Maculinea nausithous occurs on rougher types of Molinion andArrhenaterion vegetations upt o intensity agricultural useo r Agropyro-Rumicion crispi.O nwe t location and in periodically abandoned grasslands a mosaic of dry andwe t types of vegetation, areth e best habitats for both Maculinea nausithous can befound in species. In Switzerland,the y also Filipendulion, Calthionpalustris and Phragmition occur onslope s with bushes vegetations. Road verges, embankments and canal borders can bever y important as habitat. (Gonseth 1987).

Habitatpatch size and habitat use Habitat patchesfo r Maculineateleius and M.nausithous can bever y small and can havea low density of Sanguisorba officinalis. The latter butterfly species can even befoun d onsite swit h sometimes not moretha n twot othre e large,branche d plants (Gonseth 1987,GeiUle r 1990,Wynhof f

27 1992). Many sites are part of agricultural land and consist of a mosaic of abandoned and mown parts (Ebert & Rennwald 1991). Insuc hvegetation , Maculineateleius uses regrowth of Sanguisorba inth e mown partsfo r oviposition,wherea s thefemale s got oth e abandoned partsfo r nectar. Maculineanausithous tends to stay inth e abandoned partfo r aswel l ovipositioning as nectar feeding. Maculineateleius use s manyflowe r species as nectar sources whereas M.nausithous i s nearly totally restricted to Sanguisorba for feeding (Thomas 1984,Wynhof f 1992,1994) . The butterflies have asmal l home range andd o not readily leave their habitat patch (Settele 1998,Wynhof f 1995, 1996).T oa certai n extentM. nausithousmake s useo f corridors along roads,fores t edges,stream s and canals.Whil e flying over roadverges ,the y use high plants emerging fromth e vegetation canopy, sucha sflowerin g Plantago and Centaureajacea for their orientation.Th e vegetation structure along corridors seems to be important, whereas the presence of Sanguisorba probably helps indispersa l but is not really needed.Maculinea teleius ha s alowe r dispersal capacity and uses such corridors only very rarely. Inthei r normaldail y behaviour, Maculineateleius seems to be much more active than M.nausithous. The percentage of captures offlyin g individuals in mark-release-recapture studies inth e reintroduced Dutch populationwa s in allyear s remarkably higher than inMaculinea nausithous. Furthermore, in individual behaviour observations,thes e results aresupported .Ye t itwoul d bewron g toconclud e from these data that they also disperse more readily. Onth e contrary, thespecie s with more dispersal isth esomewha t "lazy" Maculinea nausithous that spends mosto f itstim e basking and resting (Wynhoff 1994, 1996).

28 Sanguisorba officinalis for nectar

Myrmica rubra Sanguisorba m-Mri MaCUUtWa officinalis as Myrmica hobt.ni t nausithous host plant I scabrinodis / /'

Vni|Rt,-ition iouc|h

Sanguisorba' officinalis and other plant species for nectar

Myrmica scabrinodis Macvlinea Sanguisorba , j M.rubra as main officinalis as j host .'int . teteius •• \ M. ruginodis host plant M. sabuleti \

Vegetation open

Figure 1.5:Venn-diagra m illustrating the complexity of interspecific relationships and the narrow niches inth e ecology of Maculinea nausithous and M. teleius.

29 Ecologyof Sanguisorba officinalis

Wet meadows with Sanguisorba officinalis arever y conspicuous during thesummer : already from adistanc e one seesth e millions of deeply red flowerheads moving inth ewind .A t aclose r lookthes e meadows belong to the plant communities Molinion and Calthion onth ewe t locations where Sanguisorba officinalisshare s its habitat with Moliniacaerulea, Succisa pratensis,Cirsium officinale and Danthonea decumbens. Ondrie r sites itca n befoun d inArrhenaterion communitie s with Filipendulaulmaria and Thalictrum flavumo n moist nutrient rich locations. Furthermore, itoccur s together withver y common plant species such as grasses alongside roads, rivers and canals.Site s with Sanguisorba have a moderate soil acidity and productivity. Duringth ewinter , locations might be inundated, but inth e summer they aredr y (Oberdorfer 1979,Weed a etal. 1987) . The distribution of Sanguisorba officinalisi scircumpola r but disjunct. The species does not occur inth e Scandinavian countries. InWester n Europe it occurs mainly inrive rvalle y systems. InTh e Netherlands these are Overijsselse Vecht and Maaswit h associated tributaries (figure 1.6) (Mennemaetal. 1985,FLORBASE-2 FFloron) . Hence inTh e Netherlands its distribution isseparate d intothre e regions: Northern Overijssel,Centra l Brabant and Central Limburg.Th e species usedt o be much morecommon , but hasdecrease d due to land drainage andfertilisatio n of agricultural land. Sanguisorba officinalisi sa n important nectar sourcefo r small butterfly species and Syrphids. It is pollinated by Syrphids, but sexual reproduction occurs only rarely because the seeds germinate and establish themselves only inope n places. Usuallyth eoccurrenc e ofsuc h places iscause d by damage toth e vegetation sod,fo r instance during mowing in rainy years whenth e soil iswet .Th e plants can persist for long periods even under unfavourable conditions through their strong roots (Weeda etal. 1987,ow n unpublished data).

30 <^

Figure 1.6: Distribution of Sanguisorba officinalis inTh e Netherlands, 1975-1999 (From: Florbase-2F, Floron Dutch Flora Conservation, Leiden).

31 Ecologyof Myrmica ants

Within Europe,th egenu s Myrmicai s common andwidesprea d (Seifert 1988, 1993). Each species lives inecologicall y distinct habitats. However, there isalway s adegre e of overlap between species and in many sites,tw o orthre e Myrmicaspecie s occur incompetitio n with eachothe r (Elmes 1982). Intabl e 1.1, anovervie w ofth ecommo n Myrmicaspecie san dthei r main habitat is presented.Myrmica's ca n befoun d from cool andwe t moorlands upt o hot and dry chalk grasslands andfro m dunes to mountainsides. Onlya limited number of species ofthi s genus are associated withMaculinea parasites.

Lifecycle of Myrmica ants All Myrmica species are polygynous; ineac h nest multiple queens canb e found, from 1 to about 100dependin g onth e species. Myrmicasabuleti an d M.scabrinodis hav equit esmal l nestswit h 1 to 3queens .Th e nestso fM. rubranormall y contain about 10queens . InJul y and August thequeen s lay many eggs. Duringth e summer, mosto f them will develop intoworkers , but laterwhe nth etemperatur e decreases,th e development will slow down.A t the end of September, a nest contains about as many hibernating larvae as workers. The larvae show somevariatio n insize .Th e bigger and heavier oneswil l develop intoyoun g queens after hibernation inth e nextspring ,whil eth e smaller oneswil l beworker s or males. Finally inJune ,whe nth equeen s start to layegg sagain ,al l larvae have reached the pupal phase.Abou t a month later, newworkers , males andvirgi n queens will accompany her. Ingeneral , unfertilised eggswil ldevelo p into males,whil e fertilised give queens or workers,dependin g onth e care andfoo d given tothem .Onl yth eworker s will leaveth e nestt o search and bring infood , the sexual antswil l stay inside untilth e nuptialflight s inth e lastweek s ofAugus t and beginning of September. Myrmica ants are rather primitive, there is nosoldie r caste (H6lldobler& Wilson 1990). Inth e contact between ants and caterpillars, an integrated set oflife - history characteristics isneeded ,an d hasevolved ,t o enlargeth echanc e ofa caterpillar to befoun d byth e right antspecie s and latero nt o optimise the useo f ant larvae as important food source.Th e periods of highest activity in Myrmicaant s are inth e early morning hours andfro m the afternoon toth e early evening.A t that time manyworker s leaveth e nestt o search for food. Thefourt h instar caterpillars leave their host plant inth e late activity period.I t

32 seems to be important tothe m to befoun d assoo na s possible after they have left the hostplant. Ifth e caterpillars havet owai t long before being found,thei r chance to beadopte d and takent oth e ant nestdecreases . Furthermore,th e other common ant species fromth egenu s Lasius and Formica, which normally also occur inth e habitats ofth e different Maculinea species, have passedth e periodo f maximum activity inth e lateafternoon . Thereafter, the chance for the caterpillar to befoun d bya predato r worker ant declines. After hibernation and pupation,th eyoun g butterflies are confronted with the presence ofthei r hosts. Rearing experiments have shown that the more butterflies eclose onth e same day,th e smaller the chance of survival isfo r the later ones.Th e ants become increasingly aggressive andwil l killth e butterflies before they have leftth e ant nest. However, most butterflies eclose and leave the nest inth e morning,befor e the ants become active. Besides, there seems to besom e spreading ineclosion .Thi s prevents moretha n one ortw o butterflies leavingth ean t nest onth esam eday . Howthi s spreading in eclosion comes about is notknown .

Table1.1: Th eMaculinea parasitesan dhabita tpreference so fth ecommo nEuropea nMyrmica specie s innorther n Europe(afte r Elmes& Thoma s 1992). Inbol dth etw ospecie stha tar esubjec to fth e presentstudy .

Myrmica species Maculinea parasite Mainhabita t

COOLAN D MOISTLOVIN GSPP . M. sulcinodis none Mountainsan dmoorland s M. ruginodis M. alcon Woods,we theathland s andbog s M. teleius M. lobicomis none Mountainsan dmoorland s WARMAN DMOIS TLOVIN G SPP. M. rubra M. nausithous Edgeso f bogs,river san dwood s M. teleius M. alcon M. limanica none Marshlands nearlake s M. vandeli none Bogsi nth eAlp s M. scabrinodis M. teleius Grasslands,bog san dmoor s M. nausithous M. alcon M. rebeli M. arion WARMAN D XEROPHILICSPP . M. rugulosa none Exposedgrassland s M. sabuleti M. arion Warm,dr ygrassland s

HOTAN DXEROPHILI CSPP . M. schencki M. rebeli Hotmountainsides , heathlands andsan ddune s M. specioides none Hotgrassland s inth enort h

33 A meadow isonl y suitable as a reintroduction sitefo r Maculinea butterflies,whe n there are many Myrmicaan t nestso fth egoo d host ant species.Thes e nests havet o be larget o preventfoo d shortagefo r the caterpillars during their periodo f parasitism (figure 1.5).A proportion ofth e ant nests needst o beclos et oth e host plant Sanguisorba officinalis to ensure that many caterpillars will befound . However, alarg e part must be located at greater distance toth e host plant to budan d recolonise empty patches after acaterpilla r has depleted a host ants' nest (Hochberg etal. 1994, Clarke etal. 1998) .

The studysite

The only possible sitefo r reintroduction of Maculinea teleius andM. nausithous butterflies inTh e Netherlands wasth e nature reserve "Moerputten", located ina centra l position ofth e country. Other nature reserves within theforme r distribution area ofM. teleiusan d M. nausithous were not acceptable for the reintroduction, either because the number and density of S.officinalis wasto o low, orth e specific Myrmicahos t ant species could not befoun d (Wynhoff 1992). The nature reserve "Moerputten" (115 hectares) issituate d atth e lowest place in itssurrounding s (figure 1.7).Th e lowest parts are lower than 2.00 m -NAP (below sea level: NAP is reference level inth e Netherlands), the meadows where this study has beendone ,ar e situated atth e outside borders at analtitud e of about 2.20 m+ NA P (Van Gerven etal. 1994) .Th e soil isa loam y sand,covere d bya peat layer,whic h is locally overlain again bysand y sediments deposited byforme r inundations.Village swer e situated on higher parts inth e landscape, butfro m about the year 1000A Dth e construction of dykes started to protect the areafro m flooding. Fromth e beginning ofth e 13thcentur y the economic importance and henceth e political power ofth e city of's Hertogenbosch increased.Therefor e plansfo r extended and better embankments and public rulesfo r dyke maintenance were implemented. However, asth e area formed apar t of the fortifications aroundth e city, inundations for military purposes usedt o occur quite regularly during the 16than d 17thcentury . Inth e 18thcentur y problems caused by highwaterlevel s increased. In 1740th ewhol e area between 'sHertogenbosch andWerkenda mwa sflooded . It resulted inth e realisation of a plant ocontro l thewate r by meanso f retention areas that could acta s basins to storewate r during highwate r levels soa st o decrease the chance

34 ,;'.'k."".'v' - ..- i-»T' • - >.... -

Figure 1.7:Historica l mapfro m Moerputten and its surrounding (1:50.000). From: Grate Historische Atlas van Nederland. of dyke breakage. Since 1766th e Moerputten is parto f the Beerze/ Baardwijkse Overlaat(=spillway ) (figure 1.8),whic h meanstha t inperiod s of highwate r levels ofth e rivers and streams within the landscape the Moerputten andth e agricultural fields and meadows in its surroundings were purposely flooded (Van derAals t 1989).Thi s happened in many winters (Stuurman& Foppen 1993).Yet , often dykes broke andvillage swer e still inundated. Theseflood s reached a mean level of4.0 0 m+NAP ,wit h a record of more than6.0 0 m+NA P in 1880(Va n derAals t 1989),whe n inth e night of 29 December the dyke nextt o Nieuwkuijk gavewa y and many people died (Van den Oord 1992,figur e 1.9). In 1904 highflood s were finally prevented by replacing the main river bedo fth e river Maas byopenin g the artificial canal Bergse Maas.

35 Dordrecht

Vlijmen Beersee nBaar d Drunen *0*-Hertogenbosch wijkseOverlaa t Nieuwkuijk ^ P^-^^ Moecputten DrongelensKanaal^^ C _^) OvughtseHeid e Loonsee nDrunens eDuiri ij fen

Breda Figure 1.8:Ma p of locations, rivers and streams mentioned inth e text.

In 1890th e railway between 's Hertogenbosch and Geertruidenberg was built.A railway bridgewa s constructed through the central parto fth e Moerputten,whic hwa s atth e same timeth e narrowest part ofth e Beerze/Baardwijkse Overlaat, and aroundth e bridge the peatwa sremoved . As itwa s still necessary to keep openth e possibility of high inundations in the retention area,th e bridge is7 m hig h and moretha n 50 mlong .Th e railway was mainly usedfo r transportation of materials and products of the leather and shoe-industry and coal until 1972,fo r people's transport only until 1950.Afte r a periodo f incidental useth e railway was closed in 1976 (Papenburg 1997).Whil e the rails have been removed,th e bridge is still present. It hasth estatu s of national industrial monument l| ••<>#fl^PWWP HW' and is assuc hprotected . Shortly after 1900th e ! 'Drongelens Kanaal'fro m Drongelen to 's Hertogenbosch was builtfo r drainage of the Figure 1.9:Far m on a hill between Vlijmen and 's Hertogenbosch while the region isfloode d (from: agricultural areas.Th e VanderAalst 1989). Baardwijkse Overloop was

36 closed. It meant anen dt oth e high winter inundation levels inth e Moerputten.Th e mean waterlevel inth ewinte r decreased to about 2.25 m+NAP ,bu tth e area was still flooded regularly. The Bossche Sloota tth e northwest andwes t side ofth e Moerputtenwa s usedt o remove thewate r surplust oth e stream Dieze. Figure 1.10:Natur e reserve Moerputten Duet o its position inth e before 1960, looking from the railway bridge in landscape,ther e must have beena Southeastern direction over the decoy to high upwardseapag eo f ground Drongelens kanaal {from: Heemkundekring Onsenoort 1959). water inth e low lying area south of the Maas, pushing up base-rich and sodium-rich water from deeper soil layers.Therefor e the groundwater levelwa sgenerall y quite highwit h about 2.00 m+NA P during summertime and moretha ntha t inth ewinter . Thewate r quality was influenced by precipitation and mixingwit h river- and stream water, mainly duringth e winter. Inthes etime s untilth e railway bridgewa s built,th ecente r ofth e Moerputten consisted of bogvegetation ,wher e local peatdigger s camet o winturf . The basisfo r the present day star-like pattern of ditches anddrain s must have been created then. The outside edges and the area around were most likely wet meadows and pastures ofth efarmer s living inth evillage s on the higher sandy ridges and ondwellin g mounds.A photograph taken before 1960give s agoo d impression of the landscape (figure 1.10) (Heemkundekring Onsenoort 1959).Th e butterfly fauna from 1900t o about 1960give s evidence for anextende d landscape with a mosaic ofwe tan ddr y places.A s many as 51specie s could befoun d then.We t meadowswer e colonised by Maculinea nausithous, M.teleius, Euphydryas aurinia, Clossiana selenean d Coenonympha tullia.I nth e heath M.alcon use dt o live, while onth edrie r meadows one could observe Heodestityrus, Pyrgus malvae and Mesoacidalia aglaya.A tfores t edges Carterocephalus palaemon,Nymphalis polychloros an d Nymphalis antiopaha dthei r breeding areas (Wynhoff 1992). Until 1964change s did not greatly affect the appearance ofth e Moerputten. Itwa s still anope n area with peatan dwe t meadows surrounded by many morewe t meadows. Onlyth e heathlands of Vughtse Heide onth e sandy soils several kilometers toth e southwer e separated byth e Drongelens Kanaal.Th e area was obviously large and

37 Figure 1.11:Th e nature reserve Moerputten in its surroundings (from: Grote TopografischeAtlas van Nederland). diverse enought osuppor t metapopulations ofth e butterfly species. During inundations, larval instarswer e not harmed or rescue siteswer e present to ensure recolonisation of empty places. From 1964on ,th e landscapewa s changed drastically bya lan d improvement project (a"ruilverkaveling" ) (Heemkundekring Onsenoort 1959). The Moerputtenwa s hydrologically isolated andth egroundwate r levelo fth e surrounding landwa sdecrease d by 1m .Th e brewery of Heineken was permitted to pump 8millio n m3wate r per yearfro m the deep soil layers ata distance of2. 5 kmeas to f Moerputten.Th evillag eo fVlijme n pumps up another 2.5 million m3pe ryea r ata distanc e of 3 kmwes t of the nature reserve (Streefkerk 1990). Itmean t that the upward seepage almoststopped . The groundwater leveldecrease d to 1.60 m+NA P inth e summer and 2.00m +NAP inth ewinter . Henceth eflooding s became restricted to nature reserve and"improved "th e land (figure 1.11).Fo r the populations of plant and animal

38 species inth enatur e reserve it implied aspatia l isolationfro mothe r populations andon e byon eth e sensitive and sedentary species disappeared (Database Dutch Butterfly Conservation). Nowadays not more than2 5 butterfly species arefound , a reduction of 50% in5 0year s (Wynhoff 1992). extremely wetwinter s only. Farmers settled inth e low-lying area around the As aconsequenc e ofth e changes inth e hydrology thevegetatio n of the Moerputtennatur e reserve is not lowan dope nan y more.Extende d bedso f Phragmites,Typha andtal l Carexspecie s surround the central lake.Aroun d these, moistforest s ofAlnus an dvariou s Salixspecie s are present nowadays wherewe t meadows usedt ooccu rwhe nth eare awa s still inagricultura luse . The nature reserve started toge t overgrown byfores t inth e 1960s (Van Engelen, pers.comm. )A t theoute r borders ofth e nature reserve, partially withinth eforest , different types ofgrassland s arefound , ofwhic hth e hay meadows with ahig h abundance of S.officinalis ar e most important as habitat of Maculinea. They have avegetatio n that isclassifie d as belonging toth e alliance Junco-Molinion, locallywit h characteristics ofArrhenaterion on dry sites,o r Filipendulion on moist sites (Ten Oever & Brongers 1994). Host ants of both Maculinea species are present.

Aimsand outline of this thesis

The aim ofthi s thesis ist o study the reintroduction in 1990o f Maculinea teleius and Maculinea nausithousint oth e nature reserve Moerputten inTh e Netherlands. Population establishment anddispersa l under natural conditions are monitored and explained. Inth e explanations, species-specific genetic composition and behaviour are incorporated.

The specific research questions to beanswere dare : (a) What areth e processes involved inth e reintroduction and establishment of bothMaculinea populations ? (b) Howdoe sth e genetic composition of reintroduced populations change under natural conditions, and how arethes e changes influenced byth e behaviour ofth especie s concerned? These questions will beexamine d forth etw o case-study species, Maculinea nausithous and Maculineateleius. Anovervie w ofth e study andth e logical connections between the partso f it isgive n infigur e 1.12.

39 To startwith , agenera l overview ofth e recent distribution andth efactor s threatening populations (of all Maculinea species) is presented inchapte r 2. This assessment illustrates the urgent needfo r conservation actions to prevent ora t least minimize further losses. It underlines that not only is more knowledge about theecolog y ofth e reintroduced species needed butals o morewor k hast o bedon eo nth e effects of conservation actions.

The Dutch populations were monitored indetai l after the reintroduction. Population size, population dynamics, distribution and dispersalwer e determined by means of yearly transect counts and mark-release-recapture studies.Thes e basic results aregive n inchapte r 3.

Thesucces s of reintroductions is influenced by species-specific behaviour, especially the dispersal and colonisation behaviour.Maculinea nausithous asth e more mobile species,show s dispersal behaviour andwil l colonise habitat patches with nestso f its host ant Myrmicarubra. Thi s butterfly species is morecapabl e of avoiding less suitable habitat patches and canthu s more successfully disperse and colonise empty habitat patches. Forfurthe r planning atth e landscape level and predictions about dispersal it is important to have more knowledge about whether ant-mediated oviposition cantak e place. Ifso , seemingly suitable locations with many Sanguisorba plants but no Myrmica ants could beavoide d for ovipostion, butth e females would rather deposit their eggs on host plants with hostant s nearby. This wasteste d ina open-ai r insectory experiment with similar plots but different ant species oneac h plot,a s described inchapte r4 .

At the reintroduction site,th e Moerputten nature reserve, Maculinea teleiusan d M.nausithous sho w aspatiall y separated distribution. Usually, the butterfly species co-occur onth e same patches within their habitat. Factors determining habitat selection atth e reintroduction sitewer e investigated and atth e sametime ,th e hypotheses resulting fromth e experimental study were tested.Withi n the population of Sanguisorba, the vegetation wasdescribe d in 1 x 1m plot s by means ofvegetatio n releves, vegetation structure measurements and measurements of several microclimatic variables. Theoccupanc y of Myrmica antswa sdetermined , too. First,ovipositio n was measured bycountin g the eggs onflowerheads . Sincethi s method only gave good results for Maculinea nausithous, we also observedfemale s of Maculinea teleiusdurin g oviposition.Thes efiel d data andth e distribution ofth e Maculineapopulation s throughout theyear s since

40 reintroduction were analysed tofin d specific parameters explaining the habitat selection of both species. The results aregive n inchapte r 5.

The distribution of both Maculineaspecie s inan d aroundth e Moerputten nature reserve is limited.A t present (2001), Maculineateleius i sstil l restrictedt oonl y one meadow,whil e Maculinea nausithousha s established three local populations, butdoe s not expand anyfurther . The reasonsfo r the limited dispersal may be,tha tth e habitat quality ofth e empty plots is not sufficient for persistent colonisation,o r that the distance between empty and colonised plots isto o larget o bridge.Th e results from studying habitat selection allowed for adetaile d analysis of colonisation and abandonment patterns inth e distribution of both butterfly species. The results are presented inchapte r 6. The population studies revealed that Decrease and loss of Maculinea teleius and M.nausithous chapter1.2 after the reintroduction, Maculinea nausithous did not settle atth e Reintroduction of Maculinea teleius and M.nausithous reintroduction habitat patch but on another. This occurred because

Population bottleneck | of ecologicalfactors . This behaviour resulted ina secon d potential bottleneck after the Genetics: Field studies: Experiment: reintroduction. Onth e populat on size other hand, Maculinea chapte r3.7 genotype • ant-mediated teleiussettle d atth e distrib ution oviposition chap er3 4 ohonotyp*: chapter 4 release-site and beha /iour ••liapter 4. 5,6 immediately increased in number and density. This species possibly did notg othroug h any Saiiijiiisoirba. Myntm.n and Maculinea in the landsaipH ct-apter5, 6. B bottleneck inadditio n to thefounde r population.I f

Conclusions for reintroduction of Maculinea species this iscorrect ,Maculinea chapter 8 teleiuswil l beexpecte d Figure 1.12: Relational diagram on the parts of this study. to show noo ronl y little change invariatio n of

41 quantitative or genetic characters. Thiswa s analysed byelectrophoresi s of allozymes. Incontrast , M.nausithous wil l be more likely tosho w greater changes compared toth e source population.Th e results ofth eallozym e electrophoresis are given inchapte r 7.

Thefina l synthesis (chapter 8)discusse s the connections between the different parts ofth estudy . Some additional data are added and conclusions aredrawn .

42 Chapter 2: Distribution and threats

The recentdistribution ofthe

EuropeanMaculinea species

Summary

The genus Maculinea isconfine d to the Palaearctic region. Four species occur from Central Europe to and/or ,whil e M. rebeli is probably restricted to Europe. Although within many Western European countries the distribution of the species is well known, there are still many questions. The exact status of some of the species is still unclear andth e amount of decline in this century is unknown. InAsi a especially within the nations of the former USSR, the distributions of species are even less well known. However, as a basis for good research and effective nature conservation, knowledge on these subjects is needed.

published as:Wynhof f 1998a, Journal of Conservation 2: 15-27.

43 Chapter 2: Distribution and threats

Introduction

All Maculinea species are restricted toth e Palaearctic regionan dthei r centre of distribution islocate d inCentra l to Eastern Europe and WestAsia . Five species are mentioned for Europe: Maculinea arionL and M. teleius Bergstrasser from France and Spaint o China;Maculinea nausithous Bergstrasser from Spaint o ,an d M.alcon Deni s &Schiff . andM. rebeliHirschke .Th edistributio n and status ofth e latter two are somewhat difficult todetermine : informe r times thetw ower e regarded to be subspecies ofM. alcons.l . andwer e rarely separated indistributio n data. Consequently, inol d literature it isofte n difficult, or even impossible todecid ewhic h species istreated .A sixthspecie s Maculineaarionides M .occur s only inChin a and Japan and its status is unclear.

Foral l Maculinea species,th e eastern boundaries ofth e distribution are poorly known,an dther e islittl e information onthei r distribution,statu s and ecology in many nations ofth eforme r USSR and neighbouring states.Som e information can befoun d in Lee (1982), Balint (1991a), Fukuda etal. (1992), Lukhtanov &Lukhtano v (1994), KopwyHOB& Top6yHO B(1995) , but much has stillt o befoun d inth efiel dan d inman y museum collections.Th e ofth e Maculinea species isstil l unclear, especially with respectt oth e eastern parto fthei r distribution. Further information dealing withth etaxonom y can befoun d inBalin t (1985, 1986, 1991ban d 1993)an d in Sibatani etal. (1994), who also examinesth e status of several rare species in EastAsia .

The main aimo fthi s study ist ogiv e arealisti c impression ofth e distribution ofth efiv e European /Wacu//nea-species.A furthe r aim ist o identify the reasonsfo r theirdecline .

Method

Mapswer edraw n onth e standard 50x50 km-UTM-gridscal e (Van Helsdingen etal. 1997)an d based on published literature, nationaldata ­ bases anddat a given byspecialists . Only post-1980-datawer e included and datatha t wereexac t enough to be presented inth e UTM-grids. Hence, "shaded areas"an dfaun a catalogues are not used inth e maps,bu t mentioned inth etext .A n overview ofth e recent distribution bycountr y is presented intabl e 2.1. Detailed information on Maculineaarion, M. teleius, an d M.nausithous can

44 Chapter 2: Distribution and threats

befoun d in newdat a sheets produced byth e European Invertebrate Survey andar e useda sbackgroun d informationfo rth e Habitats Directiveso fth e European Community (Thomas 1997,Wynhof f 1997a, 1997b). More details at regional and local levelca n befoun d inthes e data sheets.

Table 2.1. Occurenceo fMaculinea species inth e Europeancountrie s(Afte rVa nSwaa y& Warre n 1999).n , native;e ,extinct ; r, reintroduced,n* ,dat a becameavailabl eafte r paperwa spublished .

M. anon M. teleius M. nausithous M. alcon M. rebeli

Poland Czechia Slovacia Germany France Switzerland Yugoslavia The Netherlands Turkey n n Croatia n*. n Lithuania Belgium Latvia Estonia Denmark Sweden Albania Greece Finland Luxemburg UnitedKingdo m Norway Ireland Sicily Portugal

45 Chapter 2: Distribution and threats

Resultsand Discussion

Distribution All Maculinea species occur inCentra l Europe ina band reachingfro m the East of Francethroug h Germany, Switzerland,Austria , Czechia, Slovacia,th e Balkan countries andfurthe r toth e East. Except forM. rebeli, allspecie s can befoun d inth e regionstrechin g overth eforme r Russian states until Mongolia, China and Japan.However , the Northern and Southern boundaries differ perspecie s (Tolman& Lewington 1997).

Maculinea arionLinnaeu s 1758 (Figure 2.1)

All over Europe, populations of Maculinea arionhav e experienced severe declines duringth e20t h century, especially inth e northern part ofth e species' range. M.arion becam e extinct inth e Netherlands in 1964(Ta x 1989)an d inth e UK in 1979(Thoma s 1994). Itwa s also extinct in Belgium until summer 1996whe n it recolonised a location inth e South (Goffart 1997). Probably only2 populations survive in Denmark (Stoltze 1996,Nielsen ,pers . comm.). In Finland many local extinctions have occurred,wit h afe w populations surviving onglacia l outwash ridges nearth e south coast (Marttila etal. 1992).I nSweden ,stron g populations recently occurred onGotlan dan d Oland,whil e it islargel y extinct onth e mainland. Many populations have become extinct throughout Germany and northern France,althoug h in southern Germany more populations exist than presented onth e map.M. arionha s also largely disappeared from some mountain ranges,fo r example theJura . Iti sver y localised,bu t common where it occurs inth e Pyrenees, and remains widespread, buta t low densities between c. 1500m- 2000 m in the French,Italia nan dAustria nAlp s and inth e Cevennes (Thomas 1997).I n Poland,th e populations can befoun d inth e Southern part ofth e country (Buszko 1997). In Estonia it is rarewit h at maximum 20 populations (Viidalepp 1995). Ivinskis (1993) calls itcommo n for Lithuania,wher e it is restricted todr y pineforest s inth e eastern provinces.

Maculineateleius Bergstrasse r 1779 (Figure 2.2)

The distribution mapfo r M. teleiusoverestimate s the status in comparison toothe r species, ason edo to nth e map often representsjus t onepopulation . Moreover, many populations are not only small butals o isolatedfo r many generations. InFranc e M.teleius ha sonl y alimite d number of populations,mainl y in

46 Chapter 2: Distribution and threats t^fr-

the northeastern regions (Lhonore, 1992).Th e subspecies burdigalensis is probably extinct. Germany has isolated sites inth e central and southern regions (Ebert & Rennwald 1991). However, for example inth e Westerwald only 1% ofth e populations islarge ,whil e 85%ar esmal lo rver y small (<10 ind. perday ) (Fischer & Kunz 1994). InBavaria ,large r populations andeve n meta-populations ofM. teleiusca n befoun d ina number of regions (Bayerisches Landesamtfu r Umweltschutz 1995). InSwitzerlan d andAustri a the area occupied is restricted (Gonseth, pers.comm.) . The last Italian populations, located inth esouther n foothills ofth eAlps ,ar e also declining andthreatene d byth e loss of suitable habitat (Balletto 1992). InHungar y and Slowenia,M. teleius only occurs inth e northern regions (Balint 1991b,Jaksi c 1988). In Poland,M. teleiusi srare r anddeclinin g faster than M.nausithous. The only populations can befoun d inth e South (Buszko 1997).A t least some ofthe m arever y large. Finally,ther e are also populations in Lithuania (Ivinskis 1993),Czechi a (Kudrna 1994), Romania (Popescu-Gorj 1987,Rust i 1987), Bulgaria (Ganev 1985)& (Popov, pers. comm.). Muchfurthe r eastther e are sites ofthi s butterfly species in Western (Lukhtanov & Lukhtanov 1994,Woyciechowski ,pers . comm.).

Maculinea nausithousBergstrasse r 1779 (Figure 2.3)

InSpain ,M. nausithous onl y occurs inth e province of Soria, Northo f Leonan dnea r Madrida ta naltitud e 1100t o 1400m (Femandez-Rubio 1991, Munguira & Martin 1993). In Franceth e species occurs chiefly inth e Northeast, Alsace and Bourgogne (Lhonore, 1992), but often only ina limite d number of isolated populations. In Germany it is morewidel y distributed in the central part, Bavaria andth e southern regions of Eastern Germany. In Baden-Wurttemberg there are still quite a lot of populations (Ebert& Rennwald 1991),an d it alsooccur s inWesterwal d and Spessart,th e Palatinate, inth e region around Stuttgart and Bavaria. Isolated sitesca n be found inth e Eifelan d the Lower Rhine Region. Inth eforme r East-Germany it occurs only inth e southern regions and near Berlin (Weidlich & Kretschmer 1995).Th e Polish populations are mainly concentrated inth e South.I nsom e regionsth e species can bequit e common,bu t iti sstil ldeclinin g (Buszko 1997). InCzechia , it is restricted toth e northern regions (Kudrna 1994). Recordsfro m Switzerland areconcentrate d infou r regions:Neuchatel , Berner Oberland, Lake Zurich and parts ofth e RhineValle y (Gonseth 1987, SBN 1987). InAustria ,wher eM. nausithousi s rarertha n M.teleius, loca l populations were recorded inth e North and inth e southeastern parto f

47 Chapter 2: Distribution and threats

Steiermark (Reichl 1992).Th e Hungarian populations can befoun d notfa r away inth e northern andwester n regions ofthi s country (Balint 1991b, 1993).Severa l records are knownfro m Moldova (Popov, pers.comm. )an d Bulgaria (Ganev 1985). For Slovacia only old records areavailabl e (Hruby 1964). Furthermore,M. nausithous has beenfoun d in Northeast Turkey and inWester n Siberia.

Maculinea alconDeni s& Schiffermuller 1775(Figur e 2.4)

The distribution ofM. alconshow s ascattere d pattern of several disjunct areas. InDenmar k nearth ewester n coast ofth e mainland (Stoltze 1996)an d atth e utmost Southo f Sweden (Hammarstedt 1992),whic h areth e most northerly populations in Europe. InTh e Netherlands there aretw o regions with populations ofM. alconwit h most occuring inth e northern region (Tax 1989).Thi sare a isquit e close toth e populations inth e moorland of Northern Germany (Rohlfs, pers.comm.) . The Dutch populations onth e sandy soils in the southern provinces are comparably closet oth e Flemish ones. In Flanders,th ebutterfl y can befoun d inth e region northo fa n imaginary line from Antwerp to Maastricht (Maes &Va n Dyck 1996). In France, Maculineaalcon als o occurs indifferen t regions. The largest populations are knownfro m the Sarthe. Inth e Gironde region,population s are less dense,whil e they are smallest in Brittany and Contentin (Lhonore, 1992). InGerman y iti s restrictedt o several regionswher e itca n be locally quite common.Th edistributio n inCentra l and South Germany isprobabl y at least partly known,bu t is notye t published. Inth eforme r Eastern Germany, M.alcon i sextinc t (Reinhardt &Thus t 1993).Th edistributio n ofM. alconi n Spain is restricted to isolated populations in Cantabrica andth e western Sistema Iberico (Fernandez-Rubio 1991)a taltitude s upt o 1300m,spatiall y isolated from M. rebeli. InSwitzerlan d most populations can befoun d east ofth eAlp s from40 0t o 900 m(Gonset h 1987).Th e largest spot onth e map isforme d by Northeast Austria, Slovenia and Northern Kroatia. More populations occur inNorth - Hungary, South-Poland,Moldov a and Romania (Balint 1985, Buszko 1997, Popov, pers.comm.) . InNortheast-Turkey , M.alcon monticola wa s recorded (Hesselbarth ef a/. 1995).

Maculinea rebeliHirschk e 1904(Figur e 2.5)

The rarest European Macu//nea-species isM. rebeli. I ngeneral ,th e distribution ofthi s species extends from the Central Massif andth e

48 ^ Chapter 2: Distribution and threats

southeastern Alps in France andth e eastern Pyrenees inSpai nthroug h Central European mountainous areas inSwitzerland ,th e Dolomites andth e Appenines toth e east.Al l locations area t analtitud e of 1300-2000m (Higgins & Riley 1980). In France,th e majority of populations are located inth e east ofth e country (SBN 1987).Th e Spanish populations are restricted toth e central andwester n Pyrenees andth e western Sistema Iberico ataltitude s from 900 to2000 m (Fernandez-Rubio 1991).Th e distribution iscompletel y separated from M.alcon. I nSwizerlan d M.rebeli onl y occurs inmountainou s areas in the Jura andth eAlp s ataltitude s of 800t o 2300 m(Gonset h 1987). InGermany , isolated populations can befoun d in Eastern Westphalia (Meyer 1992)o nth e SwabianAlb ,an d in Bavaria (Ebert & Rennwald 1991, Doleke fal. 1998) . Inothe r mountainous areas in Germany, more populations probably occur. InEaster n Germany, M.rebeli ca nonl y been found inThuringe n (Reinhardt &Thus t 1993). InCzechia ,th e species is restricted toth efa r south (Kudrna 1994).Th e only Polish population occurs inth e Pieniny mountains (Buszko 1997).M. rebeli ha s not beenfoun d in Turkey nor inSiberia .

Status The IUCNworl d status of M. arion,M. alcon an d M.rebeli i s 'vulnerable', while M.teleius an d M.nausithous are considered to be 'endangered' (IUCN 1990, old categories). M.arion i s listed inappendi x IV, but not appendix II, of the EC Habitats Directive (Thomas 1997). Maculinea teleiusan dM. nausithous are listed in both appendeces (Wynhoff 1997a , b),whil e M. alcon and M.rebeli ar e not included.Also ,onl ythre e ofth e EuropeanMaculinea species are mentioned inth e Bern Convention: M.arion, M. teleius andM. nausithous. Inalmos t allcountrie s that havedevelope d Red Lists,on e or more ofth e Maculineaspecie s have awel ldefine d and protected status,althoug h there are many differences between countries andstate s and/or provinces (Blabet al.1984 ,Buszk o 1997,Eber t & Rennwald 1991, Gepp 1983,Gonset h 1987, Maes& Va n Dyck 1996, Munguira & Martin 1993,Wynhof f &Va n Swaay 1995).Th e relatively best protected species is M.teleius, while M.rebeli i s least protected.Thi s is probably duet o its still unclear taxonomical status. In most countries where (some of)th e species are protected by law, nature conservation stillface d the problem that habitats are notprotected .

49 Chapter 2: Distribution and threats

Reasonsfor decline

In most European countries, Maculinea species are declining. For example inTh e Netherlands four Maciv/Znea-speciesoccurre d atth e beginning ofth e century, but inth e seventies all buton e became extinct (Tax 1989).Th e reasons forth e decline are often similar to other butterfly species butth e highly specialised,myrmecophilou s Maculinea's have been especially badly affected. Most populations havedisappeare d as a result of habitat destruction and habitat deterioration. Many habitats have also been destroyed byth e development of suburbs,factories , infrastructure and recreational objects (Tax 1989,Settel e 1990,Thoma s 1991, Elmes& Thomas 1992, Munguira & Martin 1993,Thoma s 1997).Th e habitat changes as a result of changes inagricultura l techniques, such as increase in mowing frequency, drainage of patches oreve n complete regions, intensification of grazing or, alternatively, abandonment. However, many populations depend on somefor m of disturbance by man to maintainthei r habitat through traditional methods of agriculture (Thomas 1997).Thi s isshow nfo r M. arioni nth e UK:abou t 50%o f extinctions were caused byseriou s deteriorations ofth e site by intensive agriculture. The other half resulted from the abandonment of sites byfarmers , leading toa n increase invegetatio n height and cover and asubsequen t rapid losso f Myrmica sabuleti(Thoma s 1994). Insom e countries,th e decline of rabbits through myxomatosis madething s evenworse . Many populations ofM. nausithous andM. teleiusar e affected ina simila r way,eithe r by intensification of agricultural useo r byabandonmen t of sites (Ebert& Rennwald 1991, Maurin & Keith 1994).Afte r adecreas e in hostan t density duet o changes inth evegetatio n structure and inth e microclimate atth e colony sites,butterfl y numbers decrease andfinall y the populations become extinct.A t first, abandonment of meadows causes an increase in population density ofM. nausithous,bu t if continued for alonge r period,th e vegetation will become too densefo r the host ant Myrmica rubra. Under certain climatologicalcondition s and ifth e soil is notto o dryo rto owet , M.alcon an d M.rebeli population s benefit from anextensiv e grazing regime bya limited number of sheep or cattle.Withou t this regime,vegetatio n cover getsto o dense.Th e hostplant populations of Gentiana cruciatao r G. pneumonanthe are nolonge r ablet o rejuvenate and the hostan t species disappear (Kockelke ef a/. 1994). However, heavy grazing especially when theearl y larval instar caterpillars are stillo nth e hostplant, isa serious threat toth e populations (Scheper 1994).

50 C-; fxv^ Chapter 2: Distribution and threats

1. /-• '''>"&* . Ina mor e indirect way, air andwate r pollution also affect the vegetation composition and structure and this also causes deterioration in sensitive /Wactv//nea-habitats(Munguir a & Martin 1993). Habitat changes can sometimes bever y subtle,bu t nevertheless destroy suitable butterfly patches completely. At present, biotope destruction isa major threat to Maculinea populations in Mediterranean Europe and inal l eastern European Countries (Munguira & Martin 1993,Thoma s 1997, M.Woyciechowsk y pers.comm.) .

Many populations of sedentary species arethreatene d byth e fragmentation ofthei r habitats incombinatio n withth e isolation of small habitat patches. Such isolated populations arever y susceptible to chance extinction and asther e is noexchang e possible with other habitat patches, recolonisation will notoccur . Maculinea's are generally not considered to be ablet o cross adistanc e of moretha n afe w kilometers (Settele efa/ . 1996). Nowadays, many populations have already been isolated for decades and some ofthes e havegon e extinctwithou t experiencing any change inthei r habitat. Climate change may also have anegativ e effect on stenoecious, sedentary species such asth eMaculinea's. Inth e Western parto f itsdistribution ,we t heathland represents the major habitattyp e of Maculinea alcon.Durin gthi s century largearea s of heath were drained and improved for agricultural use.Th e remnants suffer from eutrophication byai r pollution.Th e vegetation changed in many cases from anope n Erica-vegetationt o aclose d one,dominate d byth e Purple moorgrass (Moliniacaerulea). There are strong indications thatth e butterfly avoidsth e larger Purple moorgrass areas. Inthi s wayfragmentatio n of populations takes place andforme r meta-populations getspli t up. InTh e Netherlands,ther e are at present only three metapopulationswhic h are capable of surviving onth e longterm .Th e other small and isolated populations appear to beth efirs t to become extinct. Even over a rather short time interval,betwee n 1980an d 1995,extinctio n of several populations was observed (Scheper 1994,Wynhof f efa/ . 1996). Insevera l cases,thi s was most likelyth e result of a lowering ofth e groundwate rtabl e andth e absence of suitable management. The lowering ofth ewate r table has restricted many populations ofGentiana pneumonanthe (and M.alcon) to the lowest (and relatively wettest) areas of the nature reserves.Thi s not only caused afragmentatio n of populations within otherwise rather continuous heathlands, but also increasedth e risko f relatively long inundations of entire gentian-populations during winters with high rainfall.Lon g periods of inundation postpone theflowerin g ofgentians , withth eeffec t that the number of budsfo r oviposition will be reduced

51 Chapter 2: Distribution and threats f'TW^p j (Wynhoff efal. 1996) .Thi s case study inTh e Netherlands isjus t anexample ; similar habitat deteriorations have occured all over Europe.

Collecting butterflies isofte n saidt o bea major reasonfo r decline.A s long as populations are large and subpopulations are connected,i t isalmos t impossible toeliminat e themjus t bycollecting . However, collectinga relatively large number of individuals could havedetrimenta l effects after habitat patches (1) havedecrease d insiz e and quality, (2) have become isolated andwhe n (3)th e butterfly numbers have already seriously decreased.

Conclusions

One or more Macu//nea-species can befoun d inth e majority of countries in Europe.M. rebeli i spossibl y restricted to Europe,th e other four occur also further east asfa r as Mongolia oreve nChin a andJapan .Unfortunately , from Finland to Italy andfro m Spaint o Poland,population s of alltreate d species aredeclinin g or have even become extinct. Meta-populations are reducedt o core-populations and core-populations vanish.Thes e processes executeda severe influence atth eedge s of distribution and inregion s thatwer e improvedfo r agriculture. Nowadays, large populations andwel l functioning meta-populations are restricted to regions with a relatively low agricultural pressure, asca n befoun d in Eastern European countries and probably inth e states ofth eforme r USSR. Inth e West European countries extinctions have occured much moreoften ,mainl y duet o intensification inagricultur e and landuse.Th e remnants get more and more isolated and suffer from a high chance of stochastic extinction. The maps (fig.2.1-2.5 ) ofth e distribution ofth e Macu/Znea-speciesgiv ea good impression ofth especies ' range. However, gaps still needt o befilled , for example mapping schemes inth e countries inth e Balkan,th eforme r USSR, France and Italywoul d add a lott o the recent knowledge onth e distribution of butterfly species. Insom e countries,dat a arealread y collected but have notye t beenpublished . Inspecie sconservatio n projects,detaile d mapso fth e local distribution should beadded .A s suchadditiona l mapsgiv eth e distribution on amor e precise scale,the y give better information onth e size,th e composition and the isolation ofth e populations. Within Europe,almos t allsite s of Maa///nea-species have been created byan d depend onsom e kind of human activity inth efor m of extensive (often traditional) agriculture to keepth evegetatio n structure openan dth ean t nest

52 ?5p, W'i?\ I Chapter 2:Distribution and threats

density sufficiently high. Nextt o habitat destruction, habitat changes asa result of improvements inagricultura l techniques form a major threat tosites . This concerns notonl y intensification ofagricultura l use,bu tals o abandonment. Therefore the protection of Maculinea populations almost always includes management of sites andthu s protection of old cultural techniques.

Acknowledgement

I want tothan k all myfriend s and colleagues whower e so kindt ogiv e me their butterfly distribution datafo r this paper: Birgit Binzenhofer, Ralf Bolz, Jaroslaw Buszko, Matthias Dolek, Roland Essayan,Konra d Fiedler, Elisabeth Geiser, Yves Gonseth, Robd eJong ,Zdravk o Kolev,Jacque s Lhonore, Dirk Maes,Christan e Meier, Gabriel Neve, PerStade lNielsen , Sergej Popov, Ole Rohlfs,Jose f Settele, KarsVeling ,mr .Voigt , Andreas Weidner, MichalWoyciechowski . Chrisva n Swaay, Janva n der Made, GerardOostermeijer , Miguel Munguira, Konrad Fiedler and an anonymous referee gave critical comments ona previous version. MartinWarre n kindly corrected the English.Pete rva n Helsdingen, European Invertebrate Survey The Netherlands, provided mewit h the map ofth e UTM-grid system and Marlies Rattink didth e computer input ofth edots .

53 fp\

£$S J& Chapter 2: Distribution and threats 'm

Figure 2.1:Distributio n of Maculinea arion in Europe.

54 •••6W Chapter 2:Distribution and threats ,s m

Figure 2.2:Distributio n of Maculinea teleius in Europe.

55 tlf Chapter 2: Distribution and threats

Figure 2.3:Distributio n of Maculinea nausithous in Europe.

56 AS.

57 $$

^ \ .-& Chapter 2: Distribution and threats ~K-h-%%t

58 Chapter 3: Population development

Populationdevelopment of the LargeBlue Butterfly species Maculineateleius and M. nausithous afterreintroduction

Summary

Details of a reintroduction of Maculinea teleius (Scarce Large Blue butterfly) and Maculinea nausithous ( Butterfly) into a nature reserve inTh e Netherlands are given.Th e introduced population of M.teleius expanded during the first three years. Incontrast , the newly established population of M. nausithous declined inth e year after the reintroduction but expanded later. After a considerable increase in numbers, this species can nowadays be seen in two subpopulations: one in and one outside the nature reserve. Although the results are quite positive with respect toth e numbers of butterflies, some alarming features have also been found. Both species, but especially M. teleius, have a very low mean minimal lifetime , which makes them more susceptible to stochastic catastrophes. While the area populated by M. nausithous isstil l expanding, M. teleius can only befoun d on the meadow where itwa s released and has not dispersed at all.Th e possible consequences of reintroductions are discussed.

Published as:Wynhof f 1998b: Lessons from the reintroduction of Maculinea teleius and M. nausithous in the Netherlands. Journal of Insect Conservation 2:47-57 .

59 Chapter 3: Population development

extinction due toth e capture of butterflies forth e reintroduction was nota risk. Furthermore,th eflowerin g period of Sanguisorba officinalisi n South Poland is parallel toth eflowerin g period inTh e Netherlands.

Habitatdescription ofthe reintroduction site The only potential sitefo r reintroduction of the Maculineabutterflie s inTh e Netherlands wasth e nature reserve "Moerputten", located ina central position ofth e country (Wynhoff 1992).Othe r nature reserves within the former distribution area ofM. teleiusan d M.nausithous wer e not acceptable for the reintroduction, either because the number anddensit y of S. officinalis wasto o low, orth e specific Myrmica host antspecie s could not befound . The nature reserve "Moerputten" (115 hectares) issituate d atth e lowest place in itssurroundings . The soil is loamy sand,covere d bya peat layer which is locally overlain by sandy sediments deposited by inundations in former times.Th e present appearance ofth e nature reserve wast o a large extent created by peat digging.Th e central lake issurrounde d bytal l bedso f Phragmites, Typha andtal l Carexspecies .Aroun d these, moistforest s of Alnusan dvariou s Salixspecie s are present nowadays wherewe t meadows usedt ooccu r whenth e areawa s still inagricultura l use.O nth e outer borders ofth e nature reserve, partially within theforest , different types of grasslands arefound ,o fwhic h the hay meadowswit h a high abundance of S.officinalis areth e most important as habitat of Maculinea. They havea vegetation that isclassifie d as belonging to the alliance Junco-Molinion, locallywit hcharacteristic s ofArrhenaterion ondr ysites ,o r Filipendulion on moist sites. Host ants of both Maculinea species are present.

Procedure ofthe Reintroduction Butterflies for reintroduction were caught with ane t andthe n placed three together ina smal l paper box.Thes e boxes were kept cool ina car - refrigerator. The butterflies were transported byca rt o The Netherlands. On thewar m evening of July 30th, 1990,a numbers of 33 malesan d 53female s ofM. teleius and 22 males and 48female s ofM. nausithouswer e released onth e meadows ofth e Moerputten.

Evaluation ofthe reintroduction

Maculinea population data The population size of both butterfly species was estimated bycapturing , marking and recapturing adults during thewhol eflight-perio d inth e Moerputten reserve and its surrounding. Ontw o meadows agri d of 10x 10

62 Chapter 3: Population development

msquare swa s permanently placed,wit h acod efo r each square. Each adult caught was marked individually bya do t code onth ewing s using aStaedle r Lumocolor Permanent pen,an d immediately released atth e point ofcapture . Foreac h capture,a recor dwa s made of sex, mark-number and grid-square code ofcapture . Outside the grid, thesit eo f capturingwa sdraw n ontoa map with a precision of about 10m . Butterflies were captured at regular intervals oftw ot othre e days.O neac h capture day, apat h of approximately the same lengthove r the meadows in the nature reserve waswalke d and inspected for adults.Walkin g speedwa s always keptth e same and thetrac kwa s neverwalke d moretha n once aday , exceptfo r the areawit h the embankment population ofM. nausithous. This area issituate d alonga smal lstrea mwit h roughvegetation ,willow s and elders where butterflies are difficult to access.Therefor e the same pathwa s walkedfou r times (without capturing butterflies moretha n one time).B y keeping thewalkin g speed constant, thetota l capture timewa s relatively short atth e beginning and end of theflight-period , but longer inth e middle when more butterflies are onth ewing .Butterflie s are assumed to havea n equal probability of capture. Ifth etota l time devoted to capture was kept constant over days,th e probability of any particular individual being captured would be inversely density-dependent: inth e optimum of theflight-perio d a lower percentage ofth e population would have been caught,whic h would have hadconsequence s for the estimation ofth e population size. Directly after the peak ofth eflight-perio d other nature reserves, meadows,roa d verges and canal embankments inth e surroundings of the "Moerputten"wer e also inspected for adults to investigate dispersal. Ifth eweathe r was badth e intervals between the investigations were occasionally longer. The reintroduced populations were studied from 1990t o 1996. In 1993,onl yM. nausithous was captured and marked,i n 1994n o mark-recapture studywa s donea tall . Theflight-perio d ofth e reintroduced population was compared to historical records offlight-period s ofth e extinct populations inTh e Netherlands andt o those ofth e source population. Data forthes e comparisons are comprized of dates offligh t recordings of individuals ofth e extant populations but consist of dates of collections mentioned oncabine t specimens.Al l data were lumped into periods offiv e dayseach .

Estimation ofpopulation size Forth e estimation of population size,tw o methodswer e used:th e Minimal Number Alive (MNA)-method (Seufert 1993) andth e method of Jolly (Begon 1979).Thi s allows for the estimation of alo w and a high limit of population

63 Chapter 3: Population development

Size. The Minimal Number Alive-method onlywork s with marked individuals, but provides noestimatio n ofth e sizeo fth e unmarked parto fth epopulation . Nextt oth edail y population size,th e mean minimal life span per captured individual can beestimated . For 1990,th etw o days of ageth e butterflies had at the moment of release,wer eadded . The method ofJoll y estimates the approximate population size over the marked and unmarked individuals (Jolly 1965). Forth e estimation the program JOLLY ofth e package CR15wa sused . Total population sizewa s calculated plotting thedail y population size estimated from theJolly - orth e MNA-analysis. The area under the curvewa s multiplied byth e mean daily lossrate .

Myrmicaan t nests The density of ant nestswa s recorded by means of inspecting plotswit ha minimum size of 2x 2 ma tdifferen t localities inth e nature reserve andth e roadverges .A s aconsequenc e of the mowing management of most grasslands,th e subterranean ant nestsar e difficult to find. They were detected bydisturbin g the soilwit h aknif e ina gri d of 10b y 10cm .Worke r antswoul d leavethei r nestwhe n itwa sdisturbed . From each nest, some individuals weretake nt oth e laboratory for species identification.

Sanguisorba officinalis Aspatia l inventory was made ofth e distribution of all Sanguisorba officinalisplant s inth e nature reserve "Moerputten"a swel l as inth e surrounding road verges. For the purposes ofthi s study, a plant was defined as being one or more stems emerging from the same place inth e soil.

Results

Populationdevelopment of M. teleiusand M. nausithous After the reintroduction in 1990th e populations of eachspecie s developed in adifferen t way. In 1991an d 1992th e number ofM. teleiusincreased , followed bya declin e toth e numbers givenfo r 1995 (figure 3.1a, 3.2a). Inth e years when noadult s have been marked,th e numbers of observed butterflies andth e counts onth e monitoring transect showtha t the populations size had been approximately the same as in 1995 (Wynhoff 1996).Th esecon d declinefro m 1995t o 1996 iscause d by accidental mowing shortly after the peakflight-perio d of the only sitewher e the

64 Chapter 3: Population development butterflies occur andovipositio n takes Mctculinea teleius place. 1990 Inth e course of the years directly •0 afterth e release,th eflight-perio d of so this species gradually shifted forwards 40 h bya fe w weeks.A statistical difference 20 inth etimin g ofth e flight-period 0 III betweenth e Moerputten andth e 1991 100 Kostrze-population can beshow n • BO (Mann-Whitney U-test:Z=-12,11 ; M K p<0,0001).Th eflight-perio d ofth e 40 indigenous populations inTh e 20 Netherlands endured untilth e endo f _jLillll.Lx_ 1992 August, withth e peak inth e lastwee k 300 of July.Th e peak population in Poland 290 200 isi nth e middle ofJul ywit hadult s l 1SO flying until late august withsom e still 100 l i being observed inSeptembe r (Figurny, 00 0 ..ililL iJ pers. comm.).Th eflight-period s ofth e 199S historical Dutch populationdiffer s from 125 that ofth e Kostre-population (Mann- 100 Whitney U-test:Z=-19,12 ; p<0,0001). 78 The Moerputten population peaksi n 90 25 thesecon d week ofJuly , andwit hth e .i ll HI » beginning ofAugust , theflight-perio d is 1996 already over. Theflight-perio d ofth e new population isdifferen t from the JO historical one (Mann-Whitney U-test: 20

10 Z=-22,53;p<0,0001).Th e li ilL... development ofth etota l population 1^4*3^5 xss size peryea r isgive n infigur e 2a.Th e total population size in 1993, 1994an d Figure 3.1a: Development in population 1995wa s about 300 individuals per size andflight-perio d of Maculinea teleius year. This is most likely the maximum inth e nature reserve Moerputten (The Netherlands) where the species had been population sizetha t can survive onth e reintroduced in 1990. single occupied meadow. Bars: minimal daily population size estimated by Minimal Number Alive Line: daily population size estimated by Jolly

65 Chapter 3: Population development

M.nausithous experienceda Afaculinect nausithous second bottleneck after thefirs t one induced byth e reintroduction. After ashar p decrease in 1991, the butterflies settled at another sitetha nth e reintroduction spot andthe n considerably increased in numbers (Figure 1b,2b) .Als o inthi s species achang e inth e flight-period compared toth e - source population can beshow n ll ii 1 (Mann-Whitney U-test:Z=-8,91 ; 1992 p<0,0001).Ther e are also r statistical differences between the historical andth e new Dutch 1 population (Mann-Whitney U-test: | | 1 .. fl1 ; Z=-12,17; p<0.001) and betweeen 1993 the historical andth e Kostrze- population (Mann-Whitney U-test: Z=-13,36; p<0,0001).Th e shift in theflight-perio d throughout the g n 11 i 9 1 years is much less compared to 1995 M.teleius (figur e 3.1a, 3.2a). The total population size peryea r sinceth e reintroduction isgive n in figure 3.2b. n 1 B il I 1 LL J s 1996 Life span - Although for the newly \ established populations of both \ - species the general tendency with - 1 J 1 . respect toth e population size is positive (i.e.increas e until Figure 3.1b: Development in population size equilibrium), the mean minimal life and flight-period of Maculinea nausithous in the time per captured individual has nature reserve Moerputten (The Netherlands) decreased strongly (figure 3.2a where the species had been reintroduced in and 3.2b). Even ifth e datafro m 1990. 1990ar e excluded astw o days Bars: minimal daily population size estimated by Minimal Number Alive under cool,dar k and predator-free Line: daily population size estimated by Jolly conditions almost certainly

66 Chapter 3: Population development

I ' ' ' i 1 1 ' ' ' i ' ' ' I o 1990 1991 1992 1993 1994 1995 1996 Year (A .2100 0 4 • £ 900 3.5 3 £ 800 3 o .»Q 70 0 3 600 2.5 z 1 ^ 2 i 500 1.5 400 1 300

200 0.5

100 Ml, -+- -H h- -+- •0 1990 1991 1992 1993 1994 1995 1996 Year

Figure 3.2:Annua l population size and mean minimal life span of (A) Maculinea teleius and (B) Maculinea nausithous in the nature reserve Moerputten (the Netherlands) where both species had been reintroduced in 1990. In 1993 only Maculinea nausithous was captured and marked, in 1994 no mark-recapture study was done. Bars: annual population size; white estimated with the Minimal Number Alive-data; grey estimated withth e Jolly data. Line: mean minimal life span estimated by MNA; square, male andfemal e adults; circle, female adults; triangle, male adults.

67 Chapter 3: Population development

increase longevity, the same conclusion must bedrawn . Inth e case ofth e female M.teleius, the mean minimal lifetim e in 1996wa sjus t one day. From all markedfemales ,onl yon ecoul d berecapture dafte r twodays .

Distribution of Maculinea teleiusand Maculinea nausithous Five years after reintroduction M. teleiusi sstil l restricted toth e single meadow inth e nature reserve,wher e the adults have been released (figure 3.3). The nest density ofth e host ant Myrmica scabrinodiswa sfoun dt o be between 0.4 and 1.3nests/m 2.Thi swa s highertha n inan yothe r parto fth e Moerputten reserve (0-0.6 nests/m2).Th e density ofth e hostplant S. officinalisi s 15plant s per m2 with onaverag e 30flowerhead s per plant. In 1991an d 1992,som e adults were observed onth e meadows atth e western border ofth e reserve; butthes e meadowswer e mown in 1992 inth e second week ofth eflight-period ,on ewee k latertha nth e proposed datejus t before thestar to fth eflight-period .Sinc ethen , lesstha n one percento fth e population has beenobserve d outside their original release meadow each year. Also, no butterflies were recorded on road verges and canal borders with similar vegetation types andstructure . M.nausithous wa s released ona meadow withtalle r vegetation and high numbers of branched S.officinalis wit h manyflowerhead s per plant. However, they desertedthi s sitet o establish a population ata neve n rougher site onth e northern sideo f the old railway embankment which runs through the nature reserve. However, the hostplant density is low. Not moretha n 30 plantswit h about 50flowerhead s per plant are available for oviposition. Since 1993,a secon d subpopulation exists onth e road verges south ofth e Moerputten.Thi ssubpopulatio n isstil l increasing and is,sinc e 1996, larger thanth efirs t population.O fal l recaptured adults,6 t o 8 %pe ryea rwa s marked inth e other subpopulation. Several individuals even migrated between thetw o sites moretha nonce . Chapter 3: Population development

Figure 3.3: Distribution of (a)Maculinea teleius and (b) Maculinea nausithous in the nature reserve "Moerputten" (The Netherlands) where both species have been reintroduced in 1990. black: Maculinea nausithous dark grey: Maculinea teleius lightgrey : Sanguisorba officinalis dot: group of Sanguisorba plants

69 Chapter 3: Population development

Discussion

Population development Populations of Maculinea butterflies are generally considered to beclose d populations. Fromcas estudie s in many parts of Europe it isknow ntha t small populations can persist onsmal l and isolated habitats for manyyears . If population size andflight-perio d between the newly established population inth e Moerputten and natural populations are compared and ifw etak e the criteria defined inth e Dutch Red Listo f Butterflies (Wynhoff &va n Swaay 1995) intoaccount ,w e can concludetha t the reintroduction wassuccessful . A population of about 300 individuals ofM. teleiuspe ryea r can evenb e considered large,a sespeciall y inthi s speciesvery ,ver y small populations have beenobserve d for many years on restricted sites (Fischer & Kunz 1994, Ebert & Rennwald 1991). However, such populations are susceptible to extinction duet o stochastic events. The last remaining indigenous Maculinea arionpopulatio n in Britainwa s of about the same size,thrivin g on a sitewit h high habitat quality. Nonetheless, extinction occurred aftera sequence offou r years when mortalities were exceptionally high dueto , amongst others, badweathe r conditions (Thomas 1995). Hence,th e number of years which a population was ablet o persist after reintroduction, isno t very reliable toevaluat e reintroduction success. The population development ofM. teleiusi nth e Moerputten is closely similar totha t ofM. arionafte r reintroduction in Britain (Thomas 1995, Thomas, pers.comm.). Both populations increased for several years,a s can beexpecte d ongoo d sites as long asther e is unlimited growth.The n numbers dropped,i n Britain through extreme drought and inTh e Netherlands through awe tan d cold summer. Anotherfacto r causing thedeclin ewa s most likely overexploitation ofth e hostan t population.Ther e are indications that the behaviour ofM. te/e/iys-caterpillars in an t nests is much liketha t ofarion- caterpillars. Both butterfly species are ablet o killof f ant colonies asa consequence of scramble competition (Thomas etal. 1993).Whe n hostant s exist insmal l colonies with high densities inth efield , it can beexpecte d that the caterpillars feed onth e broodo f more ant nests subsequently, thereby creating major damages toth e host ant population and lack offoo dfo r themselves. Eventually, evena new extinction mightfollow . Ina stabl e system,th e number, density and social structure of host ant nestswil l limit the size ofth e butterfly population, provided there is no lack of host plants.I n the HCET-modelo fth e Maculinea rebelipopulatio n of Panticosa (Spain) (Hochberg etal., 1992, 1994) butterfly numbers increased rapidly for three generations after introduction of afemal e and thendecrease d to equilibrium

70 Chapter 3: Population development

numbers,ver y much likeM. teleiusappeare d to doafte r reintroduction. It is not possible todra w such conclusions from thedevelopmen t ofth e populations ofM. nausithousafte r the reintroduction,a sth e(meta)populatio n isstil l expanding. Limitations on population growth are caused byth e small number of host plants inth e embankment population and irregular management onth e road verges-population. Upt o 20egg s perflowerhead , which isgenerall y quite large, have beenfoun d (Fiedler 1990).Thi s might lead to intraspecific competition inth eearl y larval instars.Anothe r consequence istha t too many caterpillars areadopte d byth e same host ant nest,thu sdestroyin g the nest byfeedin go nth e ant broodan d leadingt o their ownstarvation . The shift inth etimin g ofth eflight-period s shows that the phenology ofth e populations of both species have changed,probabl y as adirec t response to the new (climatical)condition s ofth e reintroduction site.Th e difference between the species' growth development ofth e populations andth e changes observed overth e study period,sugges ttha t the local adaptations might bedu et o selection onth e introduced population. Ifthi s is indeed the case,the n Irecommen d that inan yfutur e translocation asourc e population with as muchgenetica l variation as possible should beused .

Lifespan The life span of an individual isa n importantfitnes s character (Ross 1992, Stearns 1992). Itha sa grea t influence on itschanc e of successful reproduction.Thi s iseve n more important inbutterfl y species that live under limiting climatical conditions and do not migrate (Thomas etal. 1998) . Reduced longevity can bea neffec t of increased inbreeding (Orr 1994). In animal species that can bereare d inth e laboratory, data on longevity are easyt o obtain. However, both Maculinea species cannot bereare d under artificial conditions duet othei r specialised ant attendent life style,an d therefore the only possibility to estimate longevity ist ocompar e relatedfiel d parameters. Mean minimal lifetime was used,becaus e thetw o dayso fag e the butterflies werewhe nthe y were released in 1990,coul d beadded .Sinc e reintroduction,th e mean minimal lifetim e of both species has declined.Thi s decline isals o obvious,whe nth e datafro m 1990ar e excluded, because two dayso f transport under cool and dark conditions could have less impacto n the lifetim etha n twoday s inth efield . Reduced lifetim ewa s notexpected , asth e effective population size between values of 106(i n 1990)an d67 2 individuals (in 1992)fo rM. teleiusan d 96 (in 1990)an d 658 individuals (in 1995)fo r M. nausithous, calculatedfro mth ese x ratioo fth ecapture d adults, was high enought o reduceth e risko f a loss of heterozygosity (lower than

71 Chapter 3: Population development

5%: Hunter 1996),wit h the exception ofM. nausithous in 1991.I nthi s year theeffectiv e population sizewa s limitedt o8 individuals, andth e estimated risk of losso f heterozygosity was 6.25%, whereas inth eothe r years itwa s below 1%. However, mean minimal lifetim e is lower inM. teleius wher e it approaches 1da yfo rth efemales . Infiel d studies inGermany ,a mean minimal life time of 1.5day sfo r M. teleiusan d2. 1 daysfo r M. nausithous wasfoun d (Binzenhofer, pers.comm . inSettel e etal. 1996).Thes e values arethough t to bever y low, because the recapture percentage was low and duet oth e size ofth e metapopulations,th e intervals between captures were quite long. Inth e Moerputten, conditions for mark-recapture techniques were much more ideal.Probably , the lifetim e ofth eadult s is really reduced,whic h meanstha tth e populations arever y sensitivet o badweathe r periods and therefore pronet o stochastic events. Inwe t or cold summers,th efemale s cansurviv e for several dayswithou t depositing eggs. Onth efirs t available warm and sunny day,o r sunny interval between periods of rain,the y are able to lay many eggswithi n ashor t period aftime ,wherea s under optimal weather conditions,th e egg load per day ismuc h lower.

Distribution ofMaculinea teleiusand Maculinea nausithous Although many more Sanguisorba host plants and Myrmica scabrinodis ant nests can befoun d outside the single occupied meadow, M.teleius i s completely restricted in itsdistributio n toth e release site.Whil e marking the adults it happened frequently that marked specimens leftth e meadow and flew away overth e edges of thewillo wfores t or overth efiel d atth e southern border ofth e nature reserve. It might beexpecte d thatthes e butterflies were lostfo r the population but enable dispersal. However, in mostcase s when they were recaptured,i twa s onth e same meadow again,bu t notoutsid e the nature reserve, not even onth e mostsuitabl e locationswit h many host plants and Myrmica scabrinodisnests .Onl yfro m 1990t o 1992, asmal l number of adultswa sfoun d inth e meadow west ofth e occupied one.Afte r itwa s accidentally mowndurin g theflight-perio d in 1992,thi s never occurredagain . Through this event of mowing,a sever e selection pressure against mobility might have been exerted,leavin g only thever y sessile adultsfo rfurthe r successful reproduction.Althoug h all roadverge s which look suitablefo r colonisation or at least migration ofM. teleiushav e been inspected for butterflies each year, this species has never been seen outside the nature reserve onth e roadverge s and canal borders, nor in neighbouring nature reserves. Binzenhofer (pers.comm .i n Settele etal. 1996)foun d ina metapopulation inGerman y a maximum distance of2. 5 km between two observations of a marked butterfly and sheeve n calculated thatM. teleius

72 Chapter 3: Population development

was more mobiletha n M. nausithous living inth e same landscape. Hence,i t may betha t these characters have been lost byth e reintroduction. The moststrikin g aspects ofth e recent distribution ofM. nausithous since the reintroductions are (1)th efac t that they found the mostsuitabl e site in the nature reserve bythemselve s although this sitewa s considered to be unreachable duet o aban d ofwillo w and poplar vegetation,an d (2)tha tthe y did notcolonis e apparently suitable sites between the embankment andth e roadverges . During a recent inventory ofth e antfaun a in 1996 nonest s of Myrmica rubrawer e found,neithe r in most meadows of the Moerputten reserve, nor onsite s between the present subpopulations. Furthermore, whenadult s were observed outside thetw oexistin g subpopulations, resting inth evegetation ,w ewer e almost always ablet ofin d nests of its hostant . Additionally, onsite s closet oth e subpopulations, where no butterflies were observed atal lsinc e the reintroduction, noan t nests could bedetected . These observations suggest thatM. nausithousmigh t beabl et o detect and locate host ant nests.Th e mobility andth e interchange between the subpopulations arealmos tth esam ea sfoun d by Binzenhofer (pers.comm . in Settele etal. 1996) .Th e longest distance overcome bya nadul twa s more than 5k m(Binzenhofe r pers.comm .i n Settele etal. 1996 )an d included crossing landscape barriers such asagricultura l land andforests .Thes e data,togethe r withth efac t that the population aroundth e Moerputten is still expanding overabou t20 0 mo f roadverg e peryear ,give s hopefo r a positive development inth efuture .

Conclusions forfurther reintroductions

- Before starting a reintroduction of butterflies, aconsiderabl e effort should be madet o establishwhethe r thecharacteristic s ofth e release area can guarantee alon gter m survival ofth e new populations. It isals o important to payattentio nt o possibilities of dispersal and establishment ofa meta- population. - The evaluation ofth e reintroduction success should notonl y be restricted to counting or estimatingth e number of butterflies by means of monitoring transects, butals o someviabilit y characteristics, such as life span,shoul d bedetermine d regularly. - Genetical aspects should be integrated intoal l phases ofth e reintroduction procedure. It is important to select alarg e andwel l functioning source population,t otranslocat e a high number of individuals andt ofacilitat e agoo d colonisation ofth e reintroduction site by immediate increase ofth e (reproductive part ofthe )population .

73 Chapter 3: Population development

- Species-specific management isver y important tofacilitat e the colonisation. Inth e case of Maculinea butterflies,occupie d meadows and roadverge s should not be mownfro m three weeks before the start ofth e flight-period untilthre eweek s after the end ofth eflight-period . Furthermore, management shouldals o take the habitat quality ofth e host antspecie s intoaccount . However, once alarg e population has settled and does not havet ocop ewit h negative influences any more,th e management of nature reserves should concentrate onth e diversity ofth e complete system including allfaun a andflor a elements.

Acknowledgements

Iwoul d liket o express myappreciatio n to mycolleague s atth e Dutch Butterfly Conservation,th e Department of Terrestrial Ecology atth e Wageningen Agricultural University andth e Institute of Evolutionary and Ecological Sciences in Leidenfo r assistance inth efiel d and discussions on earlier versions ofth e manuscript. Furthermore Iwan t tothan k Dr. Michal Woyciechowski fromth eAgricultura l University of Krakow and hisfamil y for their hospitality and helpwit hth e parto fth estud ytha ttoo k place inPoland . The study wasfunde d byth e Netherlands Ministry ofAgriculture , Nature Conservation & Fisheries andWageninge n Agricultural University.

74 Chapter 4: Oviposition patterns

Oviposition by Maculinea nausithous and Maculineateleius butterflies inrelation to their host plant andspecific host ants

Summary

Many lycaenid butterfly species are ablet o share habitats with ants by having evolved a myrmecophilous life style:th e caterpillars provide sugary secretions for the ants and are in return protected from enemies. For obligately myrmecophilous butterflies whose caterpillars feed on ant larvae ant-related oviposition isexpecte d but was rarely found.Thi s study provides evidence for this phenomenon for the butterfly species Maculinea nausithous and Maculinea teleius. These butterflies are dependent on two sequential sources of larval food, namely the host plant Sanguisorba officinalis and for each butterfly species a different, specific, Myrmica host ant. In most occupied habitatsth e host plants are abundant while the host ant is relatively rare, so that most host plants are infac t unsuitable for larval survival due to a lack of ants inthei r close vicinity. Itwoul d give females a comparative advantage if they could detect host ants so as to deposit eggs on truly suitable Sanguisorba plants only. Oviposition in response to the density of boththes e sources offoo dwa s studied by comparing adjacent patches differing in host plant and host ant nest density. The impact of host ant presence was tested in an open air insectory experiment where females of both species were free to choose between host plants on plots with the host ant species Myrmica rubra or Myrmica scabrinodis or without ants,whil e vegetation characteristics were similar between plots. For both Maculinea species, inth e beginning ofth e flight period the presence of the respective host ant species has an influence on the oviposition of the butterflies. In the latefligh t period a heavier weighting of vegetation structure andflowerhea d phenology characters was found.

Wynhoff I, MGA van der Heijden,J G van der Made, S Plat, HHT Prins, Mva n Steenis & M Woyciechowski. Submitted.

75 Chapter 4: Oviposition patterns

Introduction

Theevolutio n of parasitic relationships iswidesprea d within manytype s of insects and other invertebrates (Begon etal. 1996 , Price 1980).Ant s represent the major parto f animal biomass intropica l climates andthe y are also important predators of smallanima l species inth etemperat e countries (Holldobler &Wilso n 1990).Therefor e they areespeciall y pronet o being parasitised. Lycaenid butterflies havesolve dth e problem of sharing habitats with ants by means of myrmecophily (Fiedler 1997). Inth e palaearctic, this concerns mostly unspecialised mutualistic relationships inwhic h butterfly larvalsecretion s provide additional foodfo rth e antswhil e the larvae benefit from protectionfro m parasitoids and predators including the antsthemselves . However, also specialised parasitism does occur. Unlike mostwester n palaearctic lycaenid species, Maculinea butterflies areobligat e parasites of ants.Afte r ashor t periodo ffeedin go nth e hostplant the larva proceeds as predator or competitor of ant brood inth e host ants' nest (Thomas 1984a, Thomas etal. 1989) . Myrmecophily can have aneffec t on many life history traits,fo r instance caterpillars offacultativ e lycaenid species grow better whentake n care of by ants (Fiedler &Saa m 1994), but also mate location,fecundity , dispersal and oviposition are affected by myrmecophily (Fiedler 1997).Survivin g ina nan t nest means living as alarv a between the risk of beingdiscovere d and predated upon ortakin g benefitfro m the dangerous host (Thomas etal. 1998).Thi s brinkmanship isespeciall y finelytone dfo r the highly specialised obligate parasite entering the brood areas ofthei r host. Inorde rt o exploit the ant nesta sefficientl y as possible, specific adaptations are needed to mimic thean t larvae inthei r behaviour and pheromones (for exampleAkin o etal. 1999). Itmean stha t the better theadaptatio n to aspecifi c host ant species has developed,th e better nests ofthi s specific antspecie s can beinvade d butals o the lower the chance ist o survive inothe r antspecies ' nests (Begon etal. 1996) . Because ofthi s and because hostselectio n carries sucha strongfitnes s component, astron g selection onovipositio n site choice is expected.Th e spatial pattern of oviposition shouldthe n overlapwit hth e spatial distribution of host ant nests at acertai n site.Caterpillar s only havea chance ofsurviva l whenth e egg has been deposited within the limited home rangeo fa Myrmica colony. Thus,a femal ewoul d increase the number of surviving offspring ifsh eoviposit s in response toth e presence ofan t nests rather than ina spatiall y randompattern . Ant-related oviposition is knownfo r some other lycaenids sucha s Plebejus argus,Ogyris amarylliso r Jalmenusevagoras. For some butterfly

76 Chapter 4: Oviposition patterns

species the pattern of distribution of oviposition isdirectl y relatedt oth e presence ofth e ants (Atsatt 1981, Pierce &Elga r 1985,Smile y era/. 1988, Jordano efa/ . 1992). Not onlyvisua l cues but alsovolatil e and non-volatile compounds can beinvolve d (Baur ef a/. 1998, Honda efal. 1997 , Henning 1983, Jutzeler 1989). However, although Maculineabutterflie s are highly specialised ant parasites, it has been claimed that thefemale s oviposit indiscriminately onan y suitable foodplant regardless whether Myrmica ants are present (Thomas 1984b,Thoma s efal. 1989) . In most habitats of Maculinea nausithous and Maculinea teleius, the hostplant Sanguisorba officinalisi sabundant ,whil e the colonies ofth e relevant hostan t species are relatively scarce on local scale (Thomas 1984). The peculiar life history strategy incombinatio n withth e patchydistributio no f Maculineanausithous and Maculineateleius population s on widespread seemingly suitable meadow habitats pleads against the random oviposition hypothesis.

HO H1

female Maculinea female Maculinea

0 + Sanguisorba [> <} Myrmica Sanguisorba -|> <] Myrmica

oviposition oviposition

numbero f numbero f oviposition oviposition

with without 1with withou t Myrmica Myrmica Myrmica Myrmica

Figure 4.1: Hypothesis of the ovipositon experiment, in which was tested whether Maculinea teleius and M. nausithous prefer to deposit their eggs in plots with their host ant species rather than in plots with other Myrmica species or without ants.

77 Chapter 4: Oviposition patterns

This study describes the impact ofth e presence of Myrmica host andnon - hostan t species onovipositio n patterns of Maculinea teleiusan d Maculinea nausithous.I ffemale s depositthei r eggs indiscriminately of the presence of Myrmicaants ,th edistributio n of observed ovipositions should not be related toabsenc eo r presence ofants . Ifthi s istru ea correlatio n with other parameters, such asfo r instance vegetation structure, may beexpected . However, iffemale s are ablet o detect antsan d respond tothei r presence, thenthi sshoul d bereveale d inth edistributio n patterns ofoviposition s (figure 4.1).Thes e alternative hypotheses were tested inth efiel d and bymean so f an insectory experiment.

Methods

Ecologyof Maculinea nausithous and M. teleius Maculinea nausithous and Maculineateleius oviposi t onflowerhead s of Sanguisorba officinalis(Thoma s 1984a, Elmes& Thoma s 1987).Th e early instar caterpillars feed ondevelopin g seeds.Afte r twot othre eweeks ,fourt h instar caterpillars leavethei r host plant to befoun d by Myrmica worker ants andtake n tothei r underground nests (figure 1.4).Th e caterpillars are obligate parasites ofth ean t nests,feedin g mainly onan t larvae. Ultimately 99% of abutterfly' s bodymass isacquire d insideth e ant nests,a swa s shown for Maculinea rebeli(Elme s etal. 1991a , b).Th e main and probably only possible host ant species for Maculinea nausithousi sMyrmica rubra, whereas Maculineateleius mainl y lives in nests of Myrmica scabrinodis (Thomas etal. 1989) . In most regions Myrmicarubra i so f minor importance tothi s latter butterfly species.Th e number of caterpillars per Myrmicanes t that develops into pupaeals o differs, primarilydependin g onth eMaculinea species concerned,bu t also on nest size, nest composition andth eMyrmica ant species involved (Elmes &Thoma s 1987;Thoma s &Wardla w 1992; Elmes etal. 1991b ;Thoma s & Elmes 1998). Inth e large nests ofMyrmica rubra,quit e a high number of Maculinea nausithous caterpillars can successfully hibernate. Incontrast , Myrmica scabrinodisha s much smaller nests inwhic h generally onlyon ecaterpilla r of Maculinea teleiusca n survive (Weidemann 1995, Bink 1992,Thoma s & Elmes 1998,ow n observations). The abundance of host ant nests is much more important thanth e abundance of host plants. However, a minimum number of host plants must grow within theforagin g ranges ofth ecolonie s ofth e host ants sotha t the caterpillar can befoun d byth eant s (Clarke etal. 1997) . Thefemale s of Maculinea teleiusprefe r very youngflowerhead s without

78 Chapter 4: Oviposition patterns

openflowers an d a high percentage offlower s still enclosed bygree n sepals. Incas eo f Maculinea nausithous, flowerheads selectedfo r oviposition are generally older and longer. Theoldes tflower s onto p are often already blooming andther e isonl ya smal l proportion stillcovere d bygree n sepals (Thomas 1984a; Figurny &Woyciechowsk i 1998). Furthermore, Maculinea teleiusdeposit s only one egg at atime ,whil e Maculinea nausithousofte n lays several eggs sequentially onth e sameflowerhead .Th e egg-load per flowerhead can be upt o 20an d more (Fiedler 1990).

Fieldstudy and experiment Inth efield , oviposition patternswer e described for different patches within the same locality. We compared these patternswit h vegetation parameters sucha sth e density of Sanguisorba officinalis, the density of both Maculinea species andth e nest densities ofthei r respective hostant sMyrmica scabrinodis and Myrmicarubra. Th e results ofth efiel d survey wereteste d in anexperimen t ina nopen-ai r insectory.Th eexperimen t consisted ofsi x plots with identical vegetationwit h Sanguisorba. Onfou r plots, Myrmica rubrao r Myrmicascabrinodis colonies were released.Maculinea females were given free choicet o deposit their eggs.Th e impact ofth e presence of hostan t species onth e oviposition behaviour ofth efemale s was studied for two periods:oviposition s duringth efirs ttw oday s anddurin gth ewhol e experimental period bycapture s of caterpillars onth e host plants.Thi sway , the oviposition behaviour inth e beginning can bedistinguishe d from later ovipositions whenth efemale s could beconfronte d witha shortag e of unoccupied flowerheads.

I Fieldwork Studyarea The area consists of aheterogenou s mosaic of abandoned and extensively managed nutrient poor, botanically rich,we t meadows inth e vicinity ofth evillag e Kostrza near Krakow (Poland) (50°15'N, 19°51'E, altitude 200m above sea level). Plant populations ofthes e meadows are dominated bycombination s of Moliniacaerulea, Festuca ovina, Trisetum flavescens, Sanguisorba officinalis, and Potentillaerecta. Th e investigated area ispar t ofth e valley of the riverWisl awit h many populations of Maculineateleius an d Maculinea nausithous. Sanguisorba officinalis, Myrmicascabrinodis and Myrmicarubra ar ecommon ,bu t densities vary between meadows (Skalski 1995,Woyciechowsk i 1991, Figurny& Woyciechowski 1998).

79 Chapter 4: Oviposition patterns

Data collection Thedensit y of ant nests (Elmes& Wardla w 1982)an d of Sanguisorba officinalis depends onvegetatio n structure and plant species composition. Withthi s inmin d2 1 homogeneous patches clearly different inthes e respects were chosen in May 1992.Al l patches were locatedwithi n an area of 1.5 km2 and ranged insiz efro m 210 m2t o 3600m 2. For each patchth efollowin g wasdetermined : 1: number of Maculinea teleiusan d M.nausithous butterflies bypoint - transect-counts twice aweek . 2: female density and number of ovipositions of Maculinea teleiusan d Maculinea nausithous. 3: density andsiz eo f Myrmica scabrinodis and Myrmicarubra nests . 4: vegetation relevesaccordin gt oth e Braun-Blanquet methodology. 5: vegetation structure measurements, including density of Sanguisorba officinalisflowerheads .

Thesedat awer e collected indifferentl y sized plotswithi nth e same homogeneous area of a patch.Th e number of ovipositions was determined forth ewhol e patch.Th edensit y of both Maculinea species ando f

300 -Maculinea teleius -Maculinea nausithous

Figure 4.2: Point transect counts of Maculinea nausithous and Maculinea teleius (males andfemales ) in 21 plots inth e valley of Kostrza (Poland).

Sanguisorba officinalis andth e vegetation composition and structure measurements were obtained ina 3 0 m2plo twithi n each patch.Nes t

80 Chapter4: Ovipositionpatterns

densities of both ant species were measured ina subplo t of 16m 2 withinth e 30 m2plot .Th e number of butterfly females entering a3 0 m2plo tove ra period of 10minute s was counted soa st o estimate densities of bothth e Maculinea species. Elevenbutterfl y countswer e done ineac h plot,twic e a week from 30Jun etil l4 Augus t 1992.Th e average ofthes e eleven counts was used as meandensity . Inthi s period both Maculinea species are abundant (figure 4.2).Th e investigations inth e patches were done during the mainfligh t periods of both species with anequa l impact of both onth e results.Th e number of ovipositions was derived from 11behaviou r protocols which describe female behaviour of each Maculinea species perpatch . Insideeac h patchth etim e neededt o locate afemal e for a next behaviour protocol wasequa lfo r every patch (5 minutes waitingtime) . The density of ant nestswa sdetermine d once,i nJun e 1992,b y completely searching the 16m 2subplot .An t nestswer e detected by pulling on plants and slightly disturbing the soilwit h aknif e at aspatia l scale of2 cm2. Per nestsom e of theworke r antstha t emerged after these disturbances were taken for identification inth e laboratory. The density of Sanguisorba officinaliswa s determined bycountin g the number of plants and the number offlowerhead s in budan d inflower . Both Maculinea species oviposit almost exclusively on buds (Thomas 1984a, Figurny &Woyciechowsk i 1998,pers . obs.). Six counts were done,weekl y from 30Jun etil l4 Augus t 1992.Th e average ofthes e six counts was used as mean density for each plot.Al lfiel d data were converted todensities .

Data analysis Thevegetatio n composition was analysed using aprincipa l component analysis (Ter Braak &Smilaue r 1998,Jongma nera/ . 1987), inwhic h samples and species are ordinated along canonical axesaccordin g to similarities inoccurrence . Plant specieswhic h were recorded injus t one plot, were excluded from analysis.A s a measure for vegetation composition,th e sample scores ofth e plotsfo r thefou r most important axeswer e takenfo r further analyses. Furthermore,th e mean Ellenberg valuesfo r humidity, productivity andacidit y were calculatedfo r eachsample . Ellenberg values are plant species specific indicator values for the releve's habitat characteristicsjus t mentioned (Kent &Coke r 1992,Ellenber g 1982). The total set of datawa s analysed bystepwis e multiple regression,wit h the oviposition density ofth e butterfly species as dependent and allothe r variables as independent values.A t theonse t of the analysis itwa s unclear whether the Maculineabutterflie s were ablet o detectthei r host ants' nests.A possibility wastha tther ewa s noan tdetectio n at all. Itwa s also possibletha t

81 Chapter 4: Oviposition patterns

antsfro m thegenu s Myrmica can bedistinguishe d from other ant genera,o r eventha tth e specific host Myrmicaspecie s withth e highest chanceo f survival forth e offspring can bedetected .Therefore ,th etota l Myrmica ant nest density was included inth e analysis, aswel l asth e density of ant colonies per species,althoug h thesetw ofactor s are not independent. Apart from this,onl y independent factors were accepted for analysis.

Insectory experiment The experiment was conducted in 1997 ina nopen-ai r gauze insectory sized4 x 12m 2.I nthi s insectory 6 plotswer e created accordingt oth e layout given infigur e 4.3. Before starting the experiment all ant nests inth e entire insectory were removed.A tth eoutsid e edges wooden shelveswit hfluo n were placedt o prevent newcolonisatio n byLasius o r Myrmicaspecie s from outside.A tth e beginning ofJune ,vegetatio n sods (40x 6 0cm )wit h meadow vegetation including Sanguisorba plants were translocated from the nature reserve Moerputten inTh e Netherlands (51°41'N,5°15'E ,altitud e 2 mabov e sea level,fo rfurthe r description seeWynhof f 1998)t oth e insectory. Each plot consisted of onaverag e 31 Sanguisorba plants (range 23-35).A t that time ofth eyea r Sanguisorba has notstarte d flowering and isstil l quite robust. Later transplantation would leadt o high mortality of plants.Al l plants andflowerhead s were marked and codedwit h small paper labels.Aroun d the plots,th evegetatio n consisted of different grass species.Thes e grasses were mown regularly. Atth e endo fJuly , nests of Myrmica scabrinodis and Myrmicarubra wer e dug out inth e nature reserve Moerputten and brought toth e insectory. The ant nestswer e placed inth e middle ofth e plots (figure 4.3) andth e antswer e giventh e possibility to settlethemselves . To control possible antmigration , pitfal ltrap s were placed atth e edges ofth e plots. Inorde r to prevent this migration,th e ant colonieswer e fed abundantly eachda ywit h wingless Drosophila (larvae and adults),frui t and sugar cubes.Th e plotswer e separatedfro m each other by3 0c mwid ewater-fille d ditches to prevent ants from leaving their home plot. Both Maculinea species prefer flowerheads of different phenological stage.A s Maculinea teleius, contraryt o Maculinea nausithous, might choose to avoid occupied flowerheads for oviposition,thi s species was released first inth e insectory. Inth e early morning ofAugus t 4th, 16youn g mated females and two males of Maculinea teleiuswer e released intoth e insectory. They were distributed equally overth e plotsan dwer e then left undisturbed. For oviposition thefemale s couldfreel y choose between 1452flowerhead s over six different plots.Afte r ashor t time,th efirs t females started to depositeggs .

82 Chapter 4: Oviposition patterns

Duringtw o days 192oviposition s were continously recorded (from9.0 0 ht o 19.00 h)an dth eflowerhead s usedwer e recorded too.Observatio n time per plotwa s kept equal. OnAugus t 8th,th e last surviving individuals of Maculinea teleiuswer e removed from the insectory. Then 16youn g mated females and 3male s of Maculinea nausithous were released and duringtw o days9 9oviposition s were recorded.Th e Sanguisorba plants andthei r flowerheads were measured.W e measured the length and height ofth e flowerheads and estimated the percentage of green, redan d flowering flowers perflowerhead . Inth e middle ofAugust ,99 5flowerhead s were encapsuled insmal l gauze bagst o prevent the caterpillars from leavingth e flowerheadthe y had lived on.Al lflowerhead s larger than 6 mm,an d asubse t ofth e smaller ones,wer e encapsuled.Thes eflowerhead s were examined daily for caterpillars.Th e caterpillars were removed and collected for other purposes, afterwhic h theflowerhea d was encapsuled again.W e recorded the butterfly speciesfoun d andth e code number of theoccupie dflowerhead . Five gauzedflowerhead s were brokendurin g handling. Directly beforeth e butterflies were released,o nAugus t 3rdon e Myrmica rubranes tfro m plot 6spli t up intotw o colonies and oneo f the sister colonies managed to enter plot 1, whichwa s meantt o bea plotwit hMyrmica scabrinodis alone.Th e Myrmicarubra colon ywa s removed immediately, but nonetheless it may have stayed onth ewron g plotfo r maximally 1.5 days. When analysing the data, it became obvious,tha tthi s Myrmica Myrmica scabrinodis short colonisation 6 1 could notb e rubra (+Myrmica neglected. rubra)

Data analysis The collected Control 5 2 Myrmica datawer e analysed rubra fortw o purposes. We investigated whether there are Myrmica 4 3 Control certain preferredo r scabrinodis rejected flowerheads for oviposition based onth e phenological 2m state andshap e of the flowerhead Figure 4.3: Layout of the open air insectory.

83 Chapter 4: Oviposition patterns

regardless of presence or absence of ants.W e assume that for an ovipositing female,th efunctiona l unitt owhic h she responds, isth e flowerhead with itscharacteristics . Therefore,a Mann-Whitney U-testwa s performed ona dat a base consisting of allflowerhead s (n=1452)t o testfo r differences between flowerheads with andwithou t eggs.Th e results show whether there are important plant characteristics, which havet o betreate d as covariables inth efurthe r analysis ofth e ant treatment. As onecolon y of Myrmica rubraha dspli t upan d invaded a ploto f Myrmica scabrinodisfou r treatments were created:tw o control plots,on e plot with Myrmica scabrinodis, two plotswit h Myrmicarubra an d onewit h both Myrmica species.Therefor e the analysiswa s conducted withth e data base consisting of allflowerheads . The antspecie s present inth e plotwa s taken asa characte r of acertai n flowerhead inth e experimental treatment. All data wereteste d for homogeneity of variances using Bartlett's test andfo r normality byth e Kolmogorov-Smirnovtes t for goodness offit .Th e assumptions for analysis ofvarianc e were repeatedly violated.Therefor e the dependence ofth e ovipositions of both butterfly species onth etreatmen t of the plotwa steste d byfittin g a linear logistic regression,usin g allth e flowerheads. Ina multiple forward logistic regression procedure,th e effects of vegetation characteristics and phenological stateo f theflowerhea d onth e oviposition were added.Fo rth e analysis ofth ecaterpilla r captures,th esam e logistic regression procedures as mentioned abovewer e repeated withth e data set of99 0gauze d flowerheads. Differences between the treatments wereteste d for significance byth e method of Tukey's honestly significant difference (after running aGLM )(Soka l& Rohlf 1995, Norusis 1990). Itcoul d bepossibl e that one ofth e plots inth e insectory would be preferred for oviposition because thevegetatio n orflowerhea d phenology characteristics werefavourabl e irrespective of the ant species present. Therefore weteste d the relative distribution ofth eflowerhea d characters overth esi x plots by means of a Kruskal-Wallis test before concentrating on the analysis ofth e oviposition behaviour. All statistical analysiswa s performed byusin gth e SPSS version 8 package (Norusis 1990, Norusis 1993).

84 Chapter 4: Oviposition patterns

Results

I Field observations Inth e Principal Component Analysis ofth e plant species composition of the plots in Poland,fou r axeswit h eigenvalues of 0.23 (axis 1),0.1 5(axi s2) , 0.12 (axis 3)an d 0.08 (axis4 ) explained 58%o fth evariatio n in vegetation species composition. Including the sample scores for thefou r major axes fromth e Principal Component Analysis intoth e multiple regression itwa s foundtha tther e was nodirec t effect of plant species composition onth e distribution ofth e Maculinea species,th e Myrmica ants orth e density of ovipositions.Th eordinatio n axes correlated positively withth e Ellenberg valuesfo r productivity (axis 1: r=0.663,p=0.001) , acidity (axis 2:r=0.748 , p<0.001) and humidity (axis 3: r=0.509,p=0.019) .Th efourt h axiswa s negatively correlated with nest size ofth e Myrmicarubra nest s (r=-0.502, p=0.021), butonl y weaklyso . The results of the multiple regression showed for Maculinea nausithous that oviposition was,withi n the measured set of parameters,onl y explained byth e density ofth e host ant species Myrmicarubra ( y =0.2 0 +2.0 6 x with y =ovipositio n density of Maculinea nausithous, x= nest density ofMyrmica rubra,standardise d regression coefficient p= 0.62 ; p= 0.003 ; r2 =0.38 )

2,5

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0,00 0,10 0,20 0,30 0,40 0,50 Myrmicarubra antnes tdensit y

Figure 4.4: The oviposition density of Maculinea nausithous in relation to the density of its specific host ant Myrmica rubra in 21 plots inth e Valley of Kostrza (Poland).

85 Chapter 4: Oviposition patterns

(figure4.4) .Nes tdensit y ofthi s antspecie s ando ffemale s ofMaculinea nausithous observed inth e plotwer e positively correlated (r=0.447, p=0.042), butthi s effect was not significant inth e regression.Th edensit y of Sanguisorba plants had nosignifican t effect onovipositio n of Maculinea nausithous.Nes t densities of both hostan t species were negatively correlated (r=-0.487,p=0.025) . Variation inth eovipositio n frequency of Maculinea teleiuswa s explained byth edensit y ofth eflowerhead s in budan d plant height (y = 1.61+ 0.00 2x ,

- 0.02 x2wit h y= ovipositio n density of Maculinea teleius, x.,= Sanguisorba

flowerheads in bud,x 2= Sanguisorba plant height, standardised regression 2 coefficients pfo) =0.53 ; p(x2)= -0.5 2 ;p = 0.001; r = 0.56) . Therewa s no effect offemal e density. Patches with many Maculineateleius females and patches withfrequen t ovipositions were notth e same.Contrar y to Maculinea nausithous,neithe r the density of antso fth e genus Myrmica north e density ofth e specific host ant Myrmicascabrinodis ha d asignifican t effect. The

Table4.1: Compariso n offlowerhead s selected and rejected for oviposition by Maculinea nausithous and Maculinea teleius.Al l flowerheads of the six plots were lumped and the presence of ants inth e plots was not included into the analyses.

Maculinea nausithous without with caterpillars caterpillars Mann •Whitney U-test N Mean Std. Dev N Mean Std. Dev. P

- flowering (%) 78 23 29 991 7 23 <0,0001 - redflower s (%) 78 45 29 991 10 19 <0,0001 - green flowers (%) 78 33 34 990 82 32 <0,0001 - height (cm) 78 97 13 1371 95 20 0,590 - length (mm) 78 13 4 1371 7 4 <0,0001

Maculinea teleius without with caterpillars caterpillars Mann -Whitney U-test N Mean Std. Dev N Mean Std. Dev. P

- flowering (%) 388 11 26 813 6 22 <0,0001 - redflower s (%) 388 21 26 813 7 18 <0,0001 - green flowers (%) 388 68 37 812 84 32 <0,0001 - height (cm) 388 96 17 1193 95 20 0,619 - length (mm) 388 10 4 1193 6 4 <0,0001

86 Chapter 4: Oviposition patterns

number of Sanguisorba flowerheads was,a sexpected ,highl y correlated both toth e number offlowerhead s in bud (r=0.917, p<0.001) andt oth e number of flowerheads in bloom (r=0.928,p<0.001) .Ther e wasals o acorrelatio n ofth e density of Sanguisorba flowerheads withth e density offemale s (r=0.588,p = 0.005). The oviposition densities of both butterfly species were independent from eachothe r (r=0.355, p=0.138).

Basedo nth e results ofth efiel dwork ,w e deduce that Maculinea nausithous might beabl et o detectth e nests of its host ant,whil e Maculinea teleiusapparentl y can noto rdoe s not usethi s information. Consequently, whenth efemale s of both species aregive nth e opportunity to deposit eggs onvegetation s similar incompositio n and structure butdifferen t inhos t ant species occupancy, eggs and caterpillars of Maculinea nausithousshoul d be foundo n plotswit hthei r hostants ,whil e those of Maculinea teleiusshoul d be distributedwit h equalfrequenc y inal lplots .

Insectory experiment Intotal , 118caterpillar s of Maculinea nausithous(7. 4 per released female) and 605 caterpillars of Maculinea teleius(37. 8 per released female) werefound . Significant differences inphenologica l stateo fth e flowerhead werefoun d between flowerheads selected for oviposition byeac ho fth e Maculinea species andth e ones rejected (table4.1) . For both Maculinea species, allcharacter s havingt o dowit hth e phenological state ofth e flowerhead,suc h asth e percentage of green, red andflowerin gflower s were important inselectin g flowerheads for oviposition. Furthermore larger flowerheads were preferred over smaller ones. Because ofthes e differences between accepted and rejected flowerheads, the proportional frequency of flowerhead characterswa s checkedfo r equal distribution overth e plots.Th e plotswer e equal,wit h the exception ofth e relative frequency distribution of thegree n coloration ofth eflowerhead s (x2=12.185, df=5,p<0.05) . Overth e plots (n=6),ther e was nocorrelatio n between the number of ovipositions or caterpillars of both butterfly species andth e number offlowerheads .

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Figure 4.5: Mean proportion of observed ovipositions and captured caterpillars with standard error of (a) Maculinea nausithous and (b) Maculinea teleius in relation to different ant treatments. cross hatched bars: ovipositions open bars: caterpillars (c) Mean proportion of green coloration per flowerhead in relation to different ant treatments. The sample sizes are asfollows : control: n=357, Myrmica rubra: n=596, Myrmica scabrinodis: n=131, M.rubra +scabrinodis: n=368. The arrows represent significantly different means (within eachtype ) (Tukey-test). Influence ofhost ants on oviposition choice Infigur e 4.5 the mean proportion of ovipositions bybot h butterfly species andth e collected caterpillars under thefou rtreatment s ispresented .Fo r Maculinea teleiusmos t ovipositions were observed onth e one plotwit hth e main host ant species Myrmica scabrinodis, butth e other ant treatments were often accepted aswell . Ovipositions of Maculinea nausithous took mainly placeo nth e plotwit h both Myrmica species.Th e plotswit hMyrmica rubraan dth e control plotswer e intermediate andth eflowerhead s inth e one with Myrmicascabrinodis wer e rarely chosen.Caterpillar s were found ina similar distribution overth e plotsfo r both butterfly species, butwit h relatively more M.teleius caterpillar s onth eflowerhead s of the plotwit h bothan t species. Logistic regressions showed significant relations between thean t treatment and the oviposition of both butterfly species. Caterpillar captureso f Maculinea teleiuswer e also significantly explained byan ttreatmen twhil eth e distribution of caterpillars of Maculinea nausithouswer e independent from it (table4.2) . Contrary to ourfindin g inth efiel d study in Poland,i nth e experiment the oviposition of both butterfly specieswer e correlated (r=0.083, p=0.002), aswel l as caterpillar captures (r=0.160, p<0.001). For the interpretation ofth e results we have to keep in mind,tha tth e number andth e phenological state ofth eflowerhead s were not equal betweenth e plotso rth etreatments .Althoug hth e number offlowerhead s per plotwa s rather different (x2=205, df=5,p<0.05) , it had no significant effect on oviposition or caterpillar density of either butterfly species.Th e percentage of green buds perflowerhea d (fig.5c )di d have asignifican t influence. For Maculinea nausithousbot hovipositio n andcaterpilla r captures were significantly affected byth e proportion ofgree n buds onth eflowerheads .A low proportion of greencoloratio n of aflowerhea d resulted in relatively more ovipositions and more caterpillars werecaptured . After incorporating flowerhead height and percentage of green budso n theflowerhea d intoth e logistic regression procedure,th eonl y significant effect onovipositio n by Maculineateleius wa s still given byan t treatment. Theeffec t ofth eant s oncaterpilla r captureswa s nowoverrule d byth e height ofth eflowerhea d inth evegetatio n and,i na negative sense, its proportion of green buds. Inth e caseo f Maculinea nausithous, thevariatio n in ovipositions aswel l as caterpillar captures was significantly explained byth eamoun t of green coloration ofth eflowerheads . The lower the percentage of green buds onth eflowerhead ,th e higher the chancestha t itwoul d beaccepte d for oviposition.Whe nth e plotwit h both Myrm/ca-specieswa s excluded from analysis,th esam e resultswer eobtained .

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Discussion

Parasites aredependen t onthei r hosts. Because of this strong dependence onthei r host, many parasites havedevelope d systems to detect the host,thu sfavourin g maximal survival ofthei r offspring. Forth e butterflies Maculinea nausithous and Maculineateleius, the hostant s onwhic h they parasitise, represent a main component ofth e habitat. Inth estudie d parto f theWisla-valle y in Poland,th e respective host antspecie s are competitors. Ingeneral ,i na vegetatio nwit h aconstan t plant species composition,onl y one Myrmica species occurs or dominates thean t community withonl y one Maculinea species parasitising upon it (Elmes efa/ . 1998).A t present, under the dynamic conditions of management styles co-existence of several Myrm/ca-speciesan d Macu//nea-species is possible. Induced bychange s in management, the Myrmicapopulation s aswel l asth e Maculineapopulation s havet o changethei r local spatial distribution overth eyear s totrac k changes inthei r habitat. Females havet o distribute about 100egg s each (estimated after Bink 1992) ina n areawit h several millions of Sanguisorba flowerheads, ofwhic h only asmal l proportion growswithi n theforagin g rangeo fth e host ants.Th e majority ofthes eflowerhead s are,thus ,sink sfo r the caterpillars. Hence only alimited ,frequentl y changing part ofth e population of hostplants willoffe r anopportunit y for survival ofth e offspring.

The useo f Sanguisorba flowerheads for Maculinea butterflies also changes withthei r phenology intim e (Thomas 1984, Figurny& Woyciechowski 1998).A flowerhea d isdurin g itsdevelopmen t atfirst a suitable egg deposition sitefo r Maculineateleius an d thenfo rMaculinea nausithous. Shortly after, itwil l beuse da sa main nectar sourcefo r both butterfly species,othe r lycaenid species and syrphids. Duet othes e changes inth e useo fth e host plant,th e local distribution patterns ofth e adults or eventh efemale s does not necessarily coincide withth eovipositio n patterns ofth especies ,a swa sfoun d by usfo r Maculinea teleiusi nth e Kostrza- meadows.Withi n thefligh t period ofth efemales , certain frequently changing locations inth e habitatwer e in use as breeding areas and others asfeedin g areas inthi s area. Inth e case of Maculinea nausithousbot hfemal e density and oviposition density were relatedt oth e density ofth e hostan tMyrmica rubra.Becaus eth eflowerhead s areolde rwhe n Maculinea nausithous deposits itseggs ,a s compared to Maculinea teleius,area s offeedin g and oviposition overlap more strongly. However, thesignifican t parameters explained 38%o fth e variance of ovipositions; 62%wa s not explained and

91 Chapter 4: Oviposition patterns

other factors aret o beidentified .

Behavioural observations show that visual cues are important inth e first phaseo fsearchin g aflowerhea dfo r oviposition.Onc ea Maculinea female hasfoun d asuitabl eflowerhead ,sh e relies onothe r cues. Forothe r butterfly species it has beenshow ntha t chemical cues can bever y important to decide whether aneg gwoul d belai d (Baur efa/ . 1998, Honda efa/ . 1997, Baylis & Pierce 1991).Th e importance ofvisua l cues isals o knownfo r the presence of ants (Jordano and others, pers.comm.) . However,Maculinea teleiusan d Maculinea nausithous do not search visuallyfo r ants.Th e oviposition behaviour does not include awal k alongth este m ofth e host plant downwards to search for ant nestso r ants. Mosto f the rather small ant nestsar e subterranean and not closet o Sanguisorba plants.So , ifth e presence of ants playsa rolefo r depositing eggs, it islikel ytha t chemical cues are involved.Fo rth e Maculinea females it could beth e scent ofth e volatile pheromoneso rth eodou r of their Myrmicahos t ant nests (Cammaerts era/. 1978, 1981; Holldobler &Wilso n 1990).

Thefiel dstud y andth e experiment give strong suggestions that there isa n impact of host ant presence onth eovipositio n byfemale s of bothMaculinea nausithous and Maculineateleius. However , while inth efiel d study Maculinea nausithousresponde d toth e presence of ant nests,i nth e experiment Maculineateleius did . Underfiel d conditions there isa high number offlowerhead s available inth efirs t periodo f egg-laying,eve n when thefemal e has very high requirements ast owhic hflowerhead s willfinall y be accepted. Later, whenth e best sites are occupied,sh e isforce d to also deposit on less suitable butstil l acceptable flowerheads. The behaviour of Maculinea teleiusi nth e insectory can beexplaine d insuc h away :Whe n analysing theearl y ovipositions only, aconvincin g effect ofth e ant treatment wasfound . When analysing caterpillar captures, vegetation structure and flowerhead phenological statewer eth e important cues.Thi stallie s withth e results ofth efiel d study in Poland.Whe n all oviposition observations during thewhol efligh t periodar e lumped,n osignifican t effect ofth eant swa sfound . The interpretation appears to betha t after having assured high chances of survivalfo r thefirs t ofthei r offspring,th efemale s prefer to deposit eggs on less suitable sites rather then stop laying eggs. Interestingly, the same phenomena have been shownfo r theovipositio n preference of Maculinea alcon,whic h exploits Myrmicanest s according toth ecockoo-strateg y (Van Dyckefa/. 2000).

92 Chapter 4: Oviposition patterns

Incas e of Maculinea nausithous, inth efiel d aclea r response toth e presence of ant nestswa s found,bu tthi swa s notfoun d inth e experiment. The majority offlowerhead s inth e experiment turned outt o be infac tto o youngfo r oviposition.Thi s meanstha t thefemale s searched for the oldest available flowerheads and apparently ratedthi s criterium higher thanth e presence of ants. This interpretation issupporte d byth e positive correlation between ovipositions of both butterfly species inth e experiment and byth e low number of eggs deposited. Indeed,th efemale s of Maculinea nausithous choset o deposit their eggs onflowerhead s which already had beenchose n byfemale s of Maculinea teleiusbefore , because these were obviously the oldest ones. Under natural conditions with asuperabundanc e of hostplants, as inth evalle y of Kostrza,ovipositio n densities of both butterfly species will not becorrelate d with each other.Afte r analysis,w etherefor e havet o conclude that inth eexperimen t for Maculinea nausithousi twa s notth e presence of ants but itwa sth eflowerhea d phenology thatwa s decisive for thefemales . Therefore the experiment should be repeated with Sanguisorba plantswit hflowerhead s ina nolde r phenological state.

Onth e contrary the results ofth efiel d study are convincingfo rMaculinea nausithous:i tshow s the highest degree of hostan t specificity among all members ofthi s genus. This Maculineaspecie s isalmos t all over Europe confined to Myrmicarubra (Thoma s ef a/. 1989bu t see Munguira &Marti n 1994).Th e advantage of deposition of eggs closet o colonies of Myrmica rubrai s related toth e spatial scale of the distribution ofth e nests.Thi s ant species tends to have large polygynous nestswit h aclumpe d distribution in the landscape.A sth eforagin g rangeo fth e ants is restricted to afe w meters aroundth e nest site, it means that the majority ofth e much more evenly distributed Sanguisorba plants are infac t unsuitable to deposit eggson . However, onean t nestca n besuccessfull y exploited bya number of caterpillars (Weidemann 1995,Eber t & Rennwald 1991, Thomas &Elme s 1998). Furthermore, Maculinea nausithousi sth e only Maculinea species which canfo r long periods survive inver y small dynamic habitats like road verges and canal borders (Ebert & Rennwald 1991). Under such conditions, itseem s natural that this rather mobile parasitic butterfly (Binzenhdfer in Settele efal. 1996 ,ow n unpublished data)ca ndetec t its host antspecies , and bythi s succesfully colonise the really suitable locations, rathertha n loose manyoffsprin g by random oviposition onsink s of Sanguisorba officinalisplant swithou t Myrmica rubra.

93 Chapter 4: Oviposition patterns

Acknowledgements Wethan k Edytha Figurnyfo r introducing ust oth efiel d site in Poland.Th e Dutch State Forestry Service kindly allowed ust otranslocat e vegetation plots and ant nestsfro m the nature reserve Moerputten. Ignas Heitkonig,Andr e Schaffers and HanOlf f gavestatistica l advice. MichielWalli s deVrie s corrected the English and Marjolein Spitteler madeth edrawings . Many thanks alsot o Chris van Swaay, Paul Lothan dJa nva n Groenendael for inspiring discussions and comments ona nearlie r draft ofthi s manuscript.

94 Chapter 5:Habitat selection

Lookingfor the ants: habitatselection oftwo Maculinea butterfliesand their Myrmica hostants

Summary

Obligate myrmecophilic butterfly species,suc h as Maculinea teleius and M. nausithous that live as a caterpillar in the nests of the ant species Myrmica scabrinodis and M.rubra, respectively, have narrowly defined habitat requirements. One would then expect that individuals are able to select for sites that meet their requirements. Both butterfly species occur in habitats where their initial larval resource,th e host plant Sanguisorba officinalis, is abundant while the ant nests are incompariso n less abundant. Inth e case of Maculinea butterflies, the presence of host ants close to the host plants determines the suitability of the host plant as being a true resource or a sink. Inth e proximity of a host plant which represents a resource, the caterpillar has a chance of being found by its host ant species,whil e inth e proximity of a sink this Myrmica species is not present and therefore the caterpillar will die. The impact of presence or absence of host ant nests on the oviposition of females and on the distribution of the population was studied on a selection of more than 600 plots in and around a nature reserve where both species were reintroduced. We found that females of both species prefer to deposit eggs on host plants inth e close vicinity of ant nests. Furthermore, adults select for plots with host ants as well. Plots with good vegetation characteristics but lack of ants were only occupied in years with high butterfly densities. We are able to reject the random oviposition hypothesis and argue that the recovery of small populations, such as after reintroductions or crashes in numbers of individuals, is supported byth e ant-mediated oviposition.

Wynhoff I, Fva n Langevelde, HOlff , M Grutters, PM Brakefield & HHT Prins.Submitted .

95 Chapter 5: Habitat selection

Introduction

Habitat selection isa phenomenon shown byanimal s that are able to move among habitats and can choose where to live. It isa result ofa combination of landscape features and behaviour ofth e .A complex form of habitat selection isth e choice of specific oviposition sites in insects (Thomas 1983a, 1983b, 1991,Bour n &Thoma s 1993, Ravenscroft 1994, Bergmann 1999, Gutierrez etal. 1999).Selectio n willfavou r individuals that use habitats inwhic h most progeny can reachth e adult stage successfully. Through this selection ecologically specialised life-history traits are generated that can make useo f this advantage (Krebs 2001, Resetarits Jr 1996).Th e morespecialise d thelif e historyo fa certain species is,th e more narrowly defined the requirements for successful development of larval instarsare . Butterflies which are specialised to parasitic larval instars are,therefore , expected to have narrowly defined requirements with respect to oviposition sites, because the choice of the oviposition site has alarg e influence onth e survival probability ofth e caterpillars. Giventhes e narrowly defined requirements, onewoul d expect that individuals are ablet o select for sites that meetthei r requirements.

The caterpillars of obligate myrmecophilebutterflie s parasitise onan t species, arar e phenomenon within the butterflies ofth e Western palearctic (Fiedler etal. 1996). In Europe,onl y butterflies ofth e genera Maculinea and Cigaritis are ablet o exploit ant nests insuc h away ,thereb y turning at leasta portion of their predators into hosts. Maculinea nausithous and Maculinea teleiusrequir e two larval resources: host plants and host ants. Both species oviposit only onflowerhead s of Sanguisorba officinalis(Thoma s 1984a, Elmes& Thoma s 1987).Th e early instar caterpillars feedo n developing seeds.Afte r twot othre e weeks,fourt h instar caterpillars leavethei r host plantt o befoun d by Myrmica worker ants andthe ntake n tothei r underground nestswher e they feed on ant larvae and hibernate. Whileth e caterpillars are adopted byan y species of Myrmica, their survival untilth e imaginalstag e depends largely onth e species of host ant.Th e mainan d probably only hostan t species suitable for Maculinea nausithousi s Myrmica rubra,wherea s Maculineateleius mainl y lives in nestso fMyrmica scabrinodis (Elmes &Thoma s 1987,Thoma s efal. 1989 ,Thoma s &Wardla w 1990, 1992, Elmes efal. 1991b ,Thoma s & Elmes 1998). Inth e caseo f Maculineabutterflies ,th e presence of host ants closet oth e host plants determines the suitability ofth e host plant asa tru e source or asink . By definition, at local level,i na sourc e the birth rate exceeds the death rate,

96 Chapter 5: Habitat selection while ina sin k the death rate exceeds the birth rate (Thomas & Kunin 1999). Inth e proximity of ahos t plantwhic h isa source ,th e caterpillar has achanc e of beingfoun d by its host ant species,whils t inth e proximity of asin kthi s Myrmica species is notpresen t andtherefor eth ecaterpilla r willdie .Thi s is especially important since Sanguisorba officinalisi softe n very abundant and widely distributed compared toth e host ants' nests.Consequently , many Sanguisorba plants are sinksfo rth e caterpillars. Ifth efemale s oviposit randomlywit h respectt o Myrmica ants as proposed byThoma s etal. (1989), Hochberg efal. (1994 ) and Clarke efal. (1997) , manyfemale s must oviposit onsinks . However, ifthe y selectfo r plotswit h host ants,the n many seemingly suitable patches that are sinks,wil l beavoided .Selectin g suitable oviposition sitesan dthereb y increasingth esurviva l probability ofth e offspring could beespeciall y important insmal l populations, sucha s in recovery from stochastic catastrophes or establishment after reintroductions. For Maculinea alcon,ther e issom e evidence that the presence of ants might beimportan t for oviposition (Scheper etal. 1995,Va n Dyck efal. 2000).Thi s has also been shownfo r Maculinea nausithous and Maculineateleius i na n insectory experiment (Wynhoff efal. 2001a), butevidenc e fromth efiel di s lacking.

We investigate the impact ofth e presence of Myrmicaant s onth e oviposition behaviour ando nth e presence of Maculinea nausithous and Maculineateleius i nth e nature reserve Moerputten (116 ha)an d its surroundings inTh e Netherlands. Inthi s area both butterfly specieswer e reintroduced in 1990 after their extinction in 1976 (Wynhoff 1998,chapte r 3). The 10year s of establishment provided uswit h datat o study the selection of habitat byth etw o butterfly species. Ourfirs t hypothesis istha t females of both butterfly species do notoviposi t randomlywit h respectt oth e presence of host ants nests.W e also useth e presence of adult butterflies asa n indicator ofthei r selectivity since adult Maculinea butterflies live onaverag ea few days (1.5t o 3day swithi n Europe) andth e majority ofthei r activity is dedicatedt othei r reproduction.Therefore ,th esecon d hypothesis istha t males andfemale s are more often found on locations with nestso fth e host anttha nwithout .

Forth eanalysis ,w edivide d the potential habitat intofou r categories. Thefirs t category includes the best habitat type: Sanguisorba grows ina n attractive vegetation andth e host ant species is present. We hypothesize that this habitat category is preferred for oviposition and isoccupie d by Maculineabutterflie s inalmos t allyears . Inth esecon d category, the

97 Chapter 5: Habitat selection

vegetation characteristics are comparable tothos e inth efirs t category, but hostant s are absent. The plots inthi s category can bedescribe d assinks . Noovipositio n isexpected . Habitat with host ants but under unfavourable vegetation conditions toth e butterflies represents thethir d category. Ifant - mediatedovipositio n takes place,the nthes e plots should beuse dfo r oviposition andcolonise d before those ofth esecon d category when butterfly density is high.Th e plotswit h inferior vegetation characteristics and lacko f Myrmica antsfal l intoth efourt h category. We expect that these plots are not usedfo r oviposition andonl y occupied inyear s with very high butterfly densities.

Methods

I Data Collection

Thefiel dwor k was carried out inth e nature reserve Moerputten (Wynhoff 1998b).Th e nature reserve issituate d inth e centre ofth e Netherlands inth e province of Northern Brabant (51°41'N,5°15'E , altitude 2 mabov e sea level,fo rfurthe r description see Wynhoff 1998b). Itconsist s of alake ,surrounde d bytal l swamp vegetation andwillo w andelde rforests . Around these,a rin g of moist meadows atth e higher locations isth e potential habitat ofth e butterflies.Th e butterflies were released onth e meadows at the southern side ofth e nature reserve. Maculineateleius colonise d the release site and expanded to afe w meadows further west. However, after onlythre e years this species was restricted toth e release site,wher e it still occurs today. Maculinea nausithousseeme d to have left the nature reserve after release, butwa sfoun d ayea r later onth e railway embankment crossing the reserve.Thi s population increased rapidly andgav e riset oth e establishment of a new local population at adistanc e of about 600 mo nth e verges of amino r road.Whil e the embankment population was rather constantfo r several years andthe n decreased in numbers,th e new local population increased rapidly (Wynhoff 1998b).Anothe r population was found two years later ata distanc e of 5kilometers .Thi sthir d population,agai n on roadverges , isstil l very small. Iti s not included inthi s study.

Sinceth e reintroduction in 1990, both Maculineateleius an d Maculinea nausithouswer e censused thoroughly. Butterflies were counted by means of transect counts and mark-release-recapture-studies (Wynhoff 1998b, Wynhoff etal. 2001b) . Furthermore, all butterfly observations during field

98 Chapter 5:Habitat selection visits were recorded ondetaile d field maps.Th e nature reserve andth e colonized roadverge swer evisite d at least once aweek , butwher e possible every secondt othir d day, during thewhol e flight period of both species. After the peak ofth efligh t period,al l roadverge s andditc h sides inth e surrounding of the Moerputten were searched for Maculineabutterflies ,too . We extracted the presence or absence of each Maculinea species per year in 1 x 1m plot sfro mth e butterfly distribution data (totalo f58 7plots) .

Maculineabutterflie s only co-occur withthei r hostplantSanguisorba officinalis. Therefore,w e restricted our detailed research tothos e parts ofth e nature reserve andth e road verges,wher e this conspicuous plant species wasfound . Inadditio n toth e host plant, Myrmica host ants are alsorequired . The composition of the local antfaun a andth e density of host ant nests depends onth evegetatio n structure ando nth e local microclimate (Elmese f al.1998) .Wit hthi s in mind,25 1 plots sized 1x 1m were chosen onal l meadows atth esouther n side ofth e nature reserve andth e roadverge s and ditch sides.W etoo k 1x 1m plots ,becaus e this size represents the scale at which afemal e decides whether to deposit aneg g or not. For each plot,th e following wasdetermined : 1: plant species composition according toth e Braun-Blanquet methodology. 2: vegetation structure measurements such as maximum height and density ofth e vegetation cover and Sanguisorba officinalisplant s and the Sanguisorba officinalisflowerhead s andthei r phenology. 3: soiltemperatur e 20c m below soil surface and mean number of sunny hours perday . All plotswer e marked inth efiel dt o enable tracing them back byth e endo f September. On33 6 additional plots,onl yth evegetatio n structure during the flight periodo fth e butterflies wasdetermined .

Per plot,w e established presence or absence of ants and determined the composition ofth e antfaun a byattractin g themwit h sugar cubes.The sugar cube represents a modelfo rth e caterpillar to befoun d byworke r ants. Inth e middleo f the plots atth efoo t of a Sanguisorba plant,a suga r cube was placedo na concav e glass plate covered with black plastic.W edi dthi s inth e early morning hours before 8o'clock , before the ants startthei r first activity period.Afte r at least one hour, the baitwa s checked forworke r ants visitingth esugar . Fromeac hspecies ,severa l antswer ecollecte dfo r identification inth e laboratory. Empty baitswer e left inth efiel d and checked later again. Inth eevening ,al l baitswer e removed.W e assume that the plots

99 Chapter 5: Habitat selection

with undetected sugar cubes represent ant-free environments atth e scaleo f our plot.

Data onovipositio n behaviour were obtained bysearchin g for deposited eggs inflowerhead s of Sanguisorba officinalis.Direc t observation of oviposition events isdifficult , because itgenerall y happens very quickly. Furthermore the eggs are deposited between theflower s onth e inflorescence ofth e host plant andar etherefor e notvisibl efro mth eoutside . Therefore,w ecollecte d flowerheads of Sanguisorba officinalisi nth e last week of September in 1997an d 1998.A t that time,th e caterpillars have left their hostplant to betake n to nestso f Myrmica antswher e they hibernate. Theflowerhead s were dried at roomtemperatur e and dissected latert o collect the eggs. Bothspecie s can beseparate d bya differen t surface pattern ofth e sculpturing onth e egg.Pe r plot, 10-30flowerhead s were collected. For Maculinea nausithous, these gave sufficient datafo r a reliable index offemal e oviposition behaviour. However, incas e of Maculinea teleius the numbero fflowerhead s collectedwa sstil lto o small incompariso nt oth e high density offlowerhead s onth eoccupie d meadow. Therefore, in 1999a restricted number offemale s were followed and observed duringoviposition . When aneg gwa s deposited,w e attracted ants inth ewa y described above and madeth e same set of measurements. A random sample of Sanguisorba plants withflowerhead s ina simila r phenological state but notaccepte d for oviposition was also studied asa control .A s it isalread y knowntha t only certain phenological states are accepted for oviposition (Thomas 1984, Figurny &Woyciechowsk i 1998),w e collected our control-sample only within flowerheads ofthi s specific character andconcentrate d onexaminin g the impact of hostant so n oviposition.Th e observations were madeo n day 14, 18,20 ,21 , 24an d 25afte r the beginning ofth efligh t period.

// Data analysis

Oviposition selection was analysed byfittin g alogisti c regression (Forward, Stepwise, Likelihood Ratio (Huisman era/. 1993)).Th e chance of finding at least oneeg g of Maculinea nausithous onon e ofth eflowerhead s in a plotwa s relatedt oth e presence ofth e host antspecies ,th e number of Sanguisorba plants andthei r number offlowerhead s andvegetatio n structure characteristics found inthi s plot. The oviposition observations ofMaculinea teleiuswer e analysed similarly.

Thevegetatio n composition wasanalyse d using a Detrended

100 Chapter 5: Habitat selection

Correspondence Analysis (Ter Braak &Smilaue r 1998,Jongma net al. 1987),i nwhic h samples andspecie s are ordinated along canonical axes according to similarities inoccurrence . Plantspecie s recorded injus t one plot were excluded from analysis.A s a measurefo r vegetation composition,th e sample scoreso fth e plotsfo rth efou r most important axeswer etake nfo r further analyses. Forth e interpretation ofth e DCA axis,w e relatedthe mt o thee log-transformedabundance s of plant species by means of stepwise linear regression. Furthermore,w e calculated the mean Ellenbergvalue s for humidity, productivity and acidity for eachsample . Ellenberg values are plant species specific indicator valuesfo r the releve's habitat characteristicsjus t mentioned (Kent &Coke r 1992, Ellenberg 1982).

After the reintroduction,ther ewer e yearswit h high and low butterfly densities. However, the number of plotso fth efirs t habitat category available forth e adults is limited.Whe nther e are many adults, it is likely that they also occupy plots ofth e second,thir d andeve nfourt h category. Therefore,w e also usedth etota l number ofoccupie d plots peryea r asa n explanatory variable. Duet oth e constant management of the nature reserve andth e roadverge s overth eyears ,w e consider thevegetatio ncomposition , vegetation structure, microclimate and antfaun a remain similar overal l years. Sanguisorba officinalisbein g a perennial plant with a root lifespano f fivet o sevenyears , isconsidere d to beconstan t innumber s of plantswhil e the number offlowerhead s per plant shows variation between theyears . Thereforew e omitted the number offlowerhead s from the statistical analysis since itwa s measured inonl y one year.

For Maculinea teleius, allyear s since the reintroduction were included inth e analysis becausethi s species immediately spread inth e nature reserve and increased in numbers. Maculinea nausithouspasse dthroug ha bottleneck in numbers in 1991,therefor e we beganth e analysis in1992 .

Thetota l seto fdat a consists of butterfly and host ant presence/absence data,th eyearl y population size ofth e butterflies,th e number ofoccupie d plots,th efou r DCAaxe s scores,vegetatio n structure and microclimate measurements. The latter three variables andth ean tdat a vary between plots butar e constant overth e years.Th e butterfly population size andth e number of occupied plots are constant overth e plots butvar y between years.Th e butterfly presence and absence data vary between both plotsan dyears .

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The presence or absence of a butterfly species was relatedt oth e data mentioned above, using logistic regression (Forward Stepwise, Likelihood Ratio (Huisman etal. 1993)).Th e resulting regression models are not intended to revealth e best possible.W e usedthe m toexplai n oviposition and distribution based onth e ecological requirements ofth e species.Therefore , wefirs t tested the influence of Myrmica hostan t presence and the number of occupied plotso nth e response variables.Then ,w e addedth e other variables tofin dth e bestfittin gmodel .

Results

Inan d aroundth e nature reserve Moerputten,th estudie d plotsar e not equalwit h respect to hostan toccurence . From64 3 plots counted for the number offlowerheads , no Myrmica antswer e attracted byth e sugar baits on 391 (=61%).Thi s meanstha tfro m 31,065 flowerheads counted only 9706 flowerheads (=31%) werewithi n reach of host ants.Thus ,21,35 9 flowerheads (=69%) were probably outo f the normalforagin g range ofth e worker ants.W efoun d Myrmica scabrinodis. on 189plot s (=29%)wit h6,53 4 flowerheads (21%).Myrmica rubra occurre d ononl y 95 plots (=15%)wit h 4,863 flowerheads (16%). Indeed,th e majority offlowerhead s ofth e hostplant Sanguisorba officinalis are sinks.Onl y asmal lfractio n ofthe m can represent apotentia l resource for the Maculinea caterpillars.

Oviposition preference Wefoun d2,24 9 eggso f Maculinea nausithous on4,40 4flowerhead s of Sanguisorba officinalis. Theflowerhead s were collected from 402 plots. Eggs were found inonl y 165o fthes e plots.O naverag e 3.2 (SD=3.1, n=365) eggs were deposited on asingl e flowerhead,wit h the highest egg loado f2 0foun d twice.W e neverfoun d eggs of one ofth e Maculinea species inth e breeding area ofth eothe r Maculinea species.

The best regression wefoun dfo rth e probability offindin g eggs of Maculinea

nausithous(p en)o na certai n plotwas :

Logit (p„) =-13.271+2.26 5 ^+2.398 T2+0.878T 3- 0.07 0T 4+0.449T 5

where T^ =presenc e of Myrmica rubra(p<0.0001) , T2= vegetatio n height e (cm) ( log-transformed)(p<0.0001) , T3= tota l number offlowerhead s of

Sanguisorba officinalis(In-transformed ) (p<0.0001),T 4= numbe r of

102 Chapter 5: Habitat selection

Sanguisorba officinalisplant s

(p<0.05)and T5= percentage ofope n soil( elog- transformed) (p<0.005).Th e model explains 82% ofth e cases correctly (N=286,- 2 Log Likelihood =200,x 2=179,df=5 , p<0.0001).Th e majority ofegg s wasfoun d on Sanguisorba plants on plotswher e the hostan tMyrmica rubrawa s also found. Whenth e Figure 5.1: Mean number of Sanguisorba officinalis hostplants (A) and flowerheads (B), proportion of plots with eggs (C) and mean factor ofant - egg load per flowerhead (D) of the butterfly Maculinea nausithous presence is in relation to the presence of the butterflies' host ant species removed from the Myrmica rubra. model,th e model Grey bars: plots with M. rubra, white bars: plots without M. rubra. was altered The number of flowerheads is equal between categories, all others are significantly different at the level of p<0.0001 (n=311). significantly witha change of 31.6 in the- 2Lo g Likelihood. Myrmicarubra appear s to bequit e rare inth e study area,sinc e it wasfoun d inonl y 25%o fth e plots usedfo r this parto fth e analysis, together with 74%o fth e plotswit h eggs (figure 5.1). When based onth e random oviposition hypothesis, 69%o fth eegg swer e expectedt o befoun do n flowerheads in plotswithou t Myrmicarubra, whil e only 26%o fth eegg s were deposited on locations without ants andca nthu s beconsidere d assinks . The number of eggs per plot issignificantl y different between plotswit ho r without Myrmica rubra.O n plotswit hth e host ant,a naverag eo f 19 (Cl(95%) = 9t o29 ) eggs inth e plotwit h 1.61(Cl(95% ) =0. 8 to 2.5) per flowerhead were deposited. Empty plots only had onaverag e 3(Cl(95% ) = 1.9 to 3.8) eggswit h 0.18 (Cl(95%) =0.1 1t o 0.24) perflowerhead .Th e number of eggs per plot (N= 323 , Mann-Whitney U= 4755 , p<0.0001) andth e egg load per

103 Chapter 5: Habitat selection

w CO 1 + ^ « E 0.8 3 C C 10 0.6 a>

0.4 -

0.2

1 0 Presence of Myrmica scabrinodis Figure 5.2: Mean proportion of ovipositions ofth e butterfly Maculinea teleius in relation to the presence of its host ant Myrmica scabrinodis. The difference between categories is significant (n=152, t=-4.310, p<0.0001). flowerhead (N =318 , Mann-Whitney U= 4503 ,p<0.0001 ) are significantly different between plotswit h andwithou t host ants (figure 5.1).

Ovipositions of Maculinea teleiuswer e morefrequentl y observed on phenologically suitable flowerheads in plotswit h ant neststha no n flowerheads inth esam e phenologically suitable state but growing in plots without ants.Th e probability that anovipositio nwil ltak e placea t a certain suitable flowerhead, is relatedt oth e presence of ants inth efollowin g way:

Logit (p.*)= -0.30 4 +1.402T 6

where T6= presenc e of Myrmica scabrinodis(p<0.0001) .Thi s model (-2 Log Likelihood =190 ,x 2=17.1, df=1, p<0.0001) explains 67%o fth e cases correctly. Apparently within the meadow where Maculineateleius occurs,al l flowerheads ina specifi c phenological stategro w under similar conditions and insimila r vegetation structure,sinc e noothe r variables could befoun dt o distinguish accepted flowerheads from arando m sample other thanth e presenceo f Myrmica scabrinodisant s (figure 5.2). Asth e presence ofthei r specific host ant isthu s shownt o be important for

104 Chapter 5:Habitat selection

oviposition in bothspecies , it is included inth e subsequent analysis ofth e distribution ofth e butterfly species.

Habitatquality description ofMaculinea teleius andMaculinea nausithous

Inth e Detrended Correspondence Analysis ofth e plant species composition ofth e plots,fou r axeswit h eigenvalues of 0.62 (axis 1), 0.28 (axis 2),0.2 3 (axis 3)an d 0.21 (axis4 )wer efound .DCA-axi s 1correlate s withth e Ellenberg productivity (r=0.887, p<0.001, n=234), Ellenberg acidity (r=0.723, p<0.001, n=229) and Ellenberg humidity (r=-0.626, p<0.001, n=236) indicator values. Nextt othese ,correlation s with some vegetation structure characteristics (vegetation height: r=0.595, Sanguisorba height: r=0.524,flowerhead s per plot:r=0.262, all p<0.0001 and numbero f Sanguisorba plants:r=-0.131, p<0.049) andsom e microclimate variables (soil temperature: r=0.253,sunshin e hours:r=-0.587 , both p<0.0001)wer e also detected.Th evegetatio n releve's areordinate d from the most nutrient poor

3,5

< U Q

Figure 5.3:Vegetatio n releves from the Moerputten nature reserve and surrounding road verges ordinated along two canonical axes. The meadows are designated BW,W H and PN, the road verges are RD and MPP,th e railroad embankment is SD. Maculinea teleius inhabits only meadow BW.Th e two populations of Maculinea nausithous are situated on RD and SD, but not on MPP,whic h is situated inth e middle of the occupied locations.

105 Chapter 5: Habitat selection

+ Carex panicea . • " .

danc e • • 5 4. • " x Cirsiumarvensi s n en • • •D o • •2 3- • Sanguisorba •

A Arrhenaterumelatio r *<> O « • a 1 • ° °<>0 AA' o Succisa pratensis 0.

-1 O Centaureajace a .10 .70 1.30 1.90 2.50 3.10 3.70

DCA axis 1 score

Figure5.4 ; Predicted abundance of some indicative plant species along DCA axis 1. meadows (BW,WH )a tth e left side infigur e 5.3 via richer meadows and road verges (RD,MPP )t o ruggedvegetation s atth e railroad embankment (SD)a t the right side infigur e 5.3.Th e DCA axisl-scores can be interpreted interm s of abundances ofth e plant species (r2=0.922, p<0.0001, F=100.3,df=25 ) in thefollowin gway :

DCA1 =1.4 9+ S p n In(abundance)fo r2 5 indicative plant species

The plant species Succisapratensis, Arrhenaterumelatior, Glechoma hederacea,Centaurea jacea, Galium mollugo,Holcus lanatus, Cirsium arvenseand Phleum pratense explaine d mosto fth evariation . Infigur e 5.4, the predicted abundance of a number ofthes e plant species and Sanguisorba officinalisi sshown .A tth e left side plant species with high abundances aretypica lfo r nutrient poor situations,whil e atth e right side species typical for more mesophilic conditions can befound . Sanguisorba officinalisha d its highest abundance inth e middleo f thefirs t ordination axis.

The probability (pt)tha t anadul t butterfly was present ina specifi c plot ina t least one ofth e study yearswa s relatedt oth e presence of its Myrmica

106 Chapter 5: Habitat selection

host species,vegetatio n height,vegetatio n composition expressed as DCA- axisl scores,an dth e number of occupied plots ina yea r (table 5.1). For both butterfly species,simila r variables gave significant effects onth e presence and absence ofth e adults. Inth e case of Maculinea nausithous, the mean Ellenbergvalu efo r humidity gave aslightl y better explanation than heighto f thevegetation . Both are correlated (n=1710, r=-0.289, p<0.01).Therefore , we decided towor k with height ofth e vegetation,sinc e this,w ethink , canb e more directly linkedt oth e behaviour ofth e butterflies.

The spatial overlap atth e scale of 1x1m 2fro m 1992t o200 0 between Maculinea nausithous and Maculineateleius i sonl y 0.66%,whil e the overlap between Myrmicarubra an d Myrmica scabrinodis wasestimate d as 3.6%. Hence except forth e presence ofth e ants,ther e are other factors important inth e habitat requirements ofth e butterfly species.Th e most important variable determining habitat selection appears to beth e DCA axisl. Maculineateleius i s restricted toth e lower ranges (figure 5.5),whil e Maculinea nausithous shows a preferencefo r higher values oftha t axis (figure 5.6). The host antspecie s show asimila r pattern alongth esam eaxis , eventhoug h less strictly separated (figure 5.7) ascompare d toth e butterfly species.

The second most important variable in habitat selection isth e presence ofth e host ant species,followe d byth e height ofth evegetation .Th e height ofth evegetatio n is notsignificantl y different between plotswit h orwithou t Myrmicarubra (t-test , p=0.636),whil e Myrmica scabrinodisi smor e frequently found inshor t vegetation (t = 10.5,p<0.001) . Inth e Moerputten nature reserve,a typica l habitat of Maculinea teleius(meado w BW) consists of an open, short vegetation upt oa height of about 50c mo na comparabl y poor soilwit h a highdensit y ofth e hostan t Myrmica scabrinodis. The host plants needt o beavailable , but high densities are not necessarily needed.Plot s with onlyfe w flowerheads are likely to beoccupied ,whil e they are notfoun d on plotswit h manyflowerhead s sincethes e sites probably have higher and more ruggedvegetation . Inyear s with high population density, the butterflies canals o beenfoun d onsuc h marginal sites. Inth esam e nature reserve, Maculinea nausithousprefer s different conditions.Th evegetatio n cover is taller and lessope ncompare d to siteswit h Maculineateleius. With respect to

107 Chapter 5: Habitat selection

Table5.1: Results of logistic regression analysis of presence/absence of Maculinea teleius or Maculinea nausithous in the nature reserve Moerputten and its surrounding.Th e regression coefficients /?and the coefficient for the intercept are given.Th e probability of observing a butterfly of one of the studied Maculinea species (M) is given by: ln[M/(1-M)] =constan t +2 p x variable.

Maculinea teleius Maculinea nausithous number of cases 1900 1710 -2 Log Likelihood 1637 552 X2 637 342 df 5 4 % predicted correct 76 94 variable S.E. Wald Sig. P S.E. Wald Sig. constant -3.750 0.395 90.092 0.000 -7.505 0.522 206.964 0.000 plotsize 0.019 0.002 76.678 0.000 0.018 0.005 14.692 0.000 DCA axis 1 4.232 0.687 37.984 0.000 1.766 0.156 128.032 0.000 (axisl)2 -3.082 0.422 53.420 0.000 Myrmica rubra 1.147 0.230 24.919 0.000 scabrinodis 0.439 0.131 11.313 0.001 vegetation height (cm) 0.027 0.011 5.788 0.016 -0.009 0.004 4.189 0.041

vegetation height there are plotswit h avegetatio n cover higher than 1 m and plotswher e thevegetatio n islowe r than about 50cm .Th efirs t category comprises plotso nth e railroad embankment (SD)an d alongside streams nextt o road verges (RD),whil e the latter category contains the roadverge s themselves (RD).Myrmica rubra ha s been captured in bothtype s of vegetation,bu t not onth ever y open nutrient poor sites inhabited by Maculinea teleius.

For both butterfly species, plotso f allfou r categories are available inth e nature reserve andth e surrounding roadverges . Category 1-plotsar e almost always occupied,whil e category 4 plotsar e empty.Th e plots of category 2 and 3sho w dynamic patterns ofoccupatio n and extinction. For Maculinea teleiusmor e potential habitat isempt y compared to Maculinea nausithous.

108 Chapter 5: Habitat selection

LU Maculinea teleius 0,6' 1*1 x> S 0,4 JiVl u 0.2" Q_ --J. •I

0.0' ^^•^•O Q O ^> Q O O 1 2 3 DCA axis 1scor e

Figure 5.5: Probability of finding adult Maculinea teleius in relation to vegetation composition expressed as DCA-axis score 1. Occupied plots are indicated with filled symbols, empty plots with open symbols.

Discussion

Randomor ant-mediated oviposition?

The results show that Maculinea nausithous and Maculineateleius can copewit hth efac t that the hostplants are very abundant andth e hostan t nests are rare,whic h meanstha t the majority of the host plants aresink sfo r the caterpillars.Amon g all Sanguisorba flowerheads ina n acceptable phenological stage,th efemale s select thosefo r oviposition thatgro w closet o a nesto fthei r host antspecies .A t the reintroduction site,fro m the apparently suitable host plants,onl y asmal lfractio n of lesstha n 30%grow s closet o Myrmicaan t nests.Thus , if random oviposition occured,a simila r fraction of the eggswoul d bedeposite d on suitable sites,whil e the restwoul d be deposited on Sanguisorba officinalisplant s that are sinks.Ou r results show that thefemale s of both species oviposit morefrequentl y inth e proximity of their host ant nests andtha t adults of both species can befoun d more frequently close to host ant nests aswell .

109 Chapter 5: Habitat selection

The resultsagre ewit hobservation s ofovipositin g females that frequently reject manyflowerhead s of several different hostplants beforethe y accept one.Clearly , the selection is not limitedt ofindin g flowerheads inth e right phenological stage (Thomas 1984,Figurn y &Woyciechowsk i 1989).Ou r study supports the results of anexperimenta l study on ant-mediated oviposition inbot hspecie s (Wynhoff etal. 2001a ,chapte r 4). Basedo nthes e findings we reject the random oviposition hypothesis.

Eventhoug h selectingfo r host plants inth e close vicinity of hostan t nests, both Maculinea species deposit a proportion ofthei r eggso nsinks . This can because d byth efac t that allflowerhead s inth evicinit y of ant nest are already occupied.Clearly , individuals are not ablet ofin d optimal oviposition siteswit h empty flowerheads close toan t nestswhe nthes e area t relatively longer distancesfro m their present position.W e hypothesize that constraints indispersa l set conditions for the selection of habitat. In Maculinea nausithous, thefractio n of eggs onflowerhead s far awayfro m ant nestswa s lesstha n in Maculinea teleius.Th e reason might betha t the egg load of Maculinea nausithousi s much higher andth efemale s areno t deterred byalread y deposited eggs (Thomas 1984).Th e nests of Myrmica rubraar e polygynous and much larger comparedt othos e ofMyrmica

Maculinea nausithous LU 0.6' 0,2' m;P

0.0 >»•«•»' ^••» *i• F 1 2 3 DCA axis 1scor e

Figure 5.6: Probability of finding adult Maculinea nausithous in relation to vegetation composition expressed as DCA-axis score 1. Occupied plots are indicated with filled symbols, empty plots with open symbols.

110 Chapter 5:Habitat selection

scabrinodis andtherefor e the number of pupae surviving inon e nest is higher. Moreover, asfa ra sonl y asmal l number ofcaterpillar s feeds onth e sameflowerhead ,competitio n forfoo d is less because the bodyweigh t ofth e caterpillar when reachingth e L4 instar is lower in M.nausithous (Thoma s 1984,Thoma s & Elmes 1998,ow n unpublished data). Hence,th e cost of increased food competition duet o laying additional eggs onth e same hostplant iscounterbalance d by higher survival chances inth e host ant nest.

Narrower restrictions toth e oviposition site characteristics as compared togenera l habitat requirements are knownfo r many butterfly species, but these restrictions are often described as limitations with regardt o microclimate and/orvegetatio n structure (Thomas 1983a,Thoma s 1983b, Thomas 1985,Warre n 1987, Sparks etal. 1994,Bergman n 1999,Gutierre z etal. 1999).Ant-relate d oviposition, however, is knownfo r myrmecophilous lycaenids intropica l regions such as Jalmenus evagorasan dothers . For some butterfly species,th e pattern of distribution of oviposition isdirectl y relatedt oth e presence of the ants (Atsatt 1981; Pierce & Elgar 1985;Smile y etal. 1988; Baylis & Pierce 1991, Jordano etal. 1992).W e also demonstrated such direct impact of host ant presence onth e oviposition of Maculinea teleius and M. nausithous.

Habitatselection inrelation to the presence ofMyrmica ants

The probability offindin g adult Maculineabutterflie s is higher on sites with certainvegetatio n characteristics and hostan t neststha n onsimila r sites where the hostant s are absent. This is notonl y due to "staying home" behaviour, but at leastwit h respect toth efemale s aresul t ofth e active searchingfo r host ant nests.Th e males spend most ofthei r lifetim e searchingfo rfemale s and are therefore also expected to befoun d closet o host ants' nests.A nextraordinaril y restricted distribution pattern despiteth e widespread distribution of the host plant is knownfo r many other butterfly species aswel l (Thomas 1991, 1995,Gutierre z etal. 1999). However, in these studies habitat sitesar e unoccupied because thevegetatio n structure around host plants is unsuitable. For both Maculinea nausithousan dM. teleius,plot s areavailabl ewhic h havesuitabl e habitatwit h respectt o vegetation characteristics butwher e the ants are missing (category 2plots) . The majority ofthes e plots are not occupied by butterflies aswell , even thoughth eadult s are potentially ablet o reachth e plots (Settele 1998). Inth e Moerputten nature reserve,severa l marked individuals of Maculinea nausithoushav e been captured in both local populations atth e railroad

111 Chapter 5: Habitat selection embankment andth e roadverges . Halfo fth estudie d meadows are situated between these twosite s and must have been crossed byth e dispersing adults. Eventhoug h these meadows have highdensitie s of Sanguisorba officinalis and,a t least locally, seemt o besuitabl efo r both Myrmicarubra an d its parasite,the ywer e notcolonise d neither were ovipositions observed.W e arguetha t this is mainly duet oth e lack of host ants nests (category 2plots) . The results also indirectly support earlier findings that Maculinea nausithous hasth e highest degree of host antspecificit y with almost entirelyMyrmica rubrabein g parasitised.Al l other Maculinea species can survive inth e nests ofsevera lofte n closely related Myrmica speciesa tth esam e sitean dwithi n different regions (Thomas ef a/. 1989, Elmes etal. 199 4bu tse e Munguira& Martin 1994). Maculinea nausithoushowever , seems to avoid sites with suitable vegetation butwit h Myrmicascabrinodis instea d of itstru e hostan t species Myrmica rubra.

The mortality of acaterpilla r before entering the Myrmicanes t has been estimated as upt o40 %(Thoma s etal. 1998 ,ow n unpublished data: Maculinea teleius50 %an dfo r Maculinea nausithous47%) .O f allth e caterpillars whichente r aMyrmica nest ,abou t 80t o 90%wil ldi e before reaching pupation (Thomas era/. 1998).Onl y after hibernation isth e mortality

1,0

T I T 9

CD O ,6- it * cp c CO

M. rubra T

DCA axis 1scor e Figure 5.7: Proportion of plots with the host ants species Myrmica rubra (filled circle) and Myrmica scabrinodis (open circle) in relation to vegetation composition expressed as DCA-axis score 1.

112 Chapter 5:Habitat selection

quite lowwit h mosto fth e surviving caterpillars being ablet o pupate ifther e is enoughfoo d available (Thomas etal. 1998, Elfferich 1998,unpublishe d data of Figurny and IW).Th e reintroduction of both butterfly specieswa s started withth e release of about 50female s that were at leasttw o daysold . Ifw e assume anequa l egg production of 50egg s perfemal e and calculateth e size ofth e population inth eyea r after reintroduction asoptimisti c as possible (70% mortality through sinks,40 %mortalit y until adoption,80 %mortalit y in ant nest),the n it isalmos t impossible toonl y double the number of translocated individuals. Surprisingly, inth e initialyear s after reintroduction, butterfly numbers increasedfour-fol d from oneyea rt oth e next.Thi s reinforces ourfindin g that oviposition inthes e butterfly species isant - mediated.

Consequences forco-occurence

Maculinea nausithousan d M.teleius ca n occur onth esam e location with high densities and apparently thriving populations (Elmes &Thoma s 1987, Ebert & Rennwald 1991, Figurny &Woyciechowsk i 1998,Settel e 1998, Wynhoff 1998a).A t such locations,th e Myrmica ant species also co-occur. Consequently, when afemal e of a Maculinea species selects aflowerhea d on a plantclos et o her hostants ' nest, other Myrmica species couldals o have their nestswithi n ashor t distance. The caterpillar behaves quite passively after leaving its hostplant. Itwil l beadopte d byth efirs t worker anttha t finds it, irrespective ofth e species.Whe n it istake n byth ewron g ant species, itwil l have a higher probability of later death inth e ant nest.Whe n spatialco - occurence of both butterfly and host ant species increases (thedistributio n of Myrmica rubraan d M.scabrinodis show moreoverlap )the na larger proportion of caterpillars of both butterfly species istake n toth ewron g ant nest.W e hypothesize thatwhe n the spatial co-occurence of hostan t nests increases,th e probability of acaterpilla r to beadopte d by its specific host ant decreases andtherefor e its mortality increases. Thiseffec t is positively correlated toth edensitie s of butterfly and host ant nests. Furthermore, it is expectedt o have more impacto nMaculinea teleius a s its hostan tMyrmica scabrinodisha s small nests inwhic h generally only one caterpillar isabl et o survive.

The advantage of deposition of eggs close to colonies ofth e host Myrmicaspecie s isals o relatedt oth e spatial scale ofth e distribution ofth e host plants. Sanguisorba officinalisi susuall yver y abundant atsite swit h Maculinea teleiusan dt o alesse r extent at siteswit h Maculinea nausithous. In

113 Chapter 5: Habitat selection

contrast, Maculinea alconan d Maculinea rebelirequir e locally distributed host plant species that areofte n rare: Gentiana pneumonanthe, G. asclepiadea and G.cruciata. I nthei r habitats,th e host plant isles s abundant thanthei r host ant species (Ebert & Rennwald 1991, Hochberg etal. 1992, 1994,Va n Dyck efal. 2000). Insuc h circumstances, a relatively low proportion of host plants represent sinks duet o lacking of host ants.Yet ,ther e are indications for host ant mediated oviposition in Maculinea alcon(Schepe r efal. 1995 , Van Dyck efal. 2000). Inthei r spatial model,Clark e efal. (1997 )an d Hochberg efal. (1994 ) assume random oviposition for Maculinea rebeli. For this species,th e conclusions drawnwoul d not bedifferen t when ant-mediated oviposition isassume d duet oth e incompariso n limited number of host plants inth e system.However ,whe n havingt ocop ewit hth esituatio ntha t most host plants are sinks, it becomes important toselec t between the ones growing in the limited home ranges ofth e host ant nests and the ones growingoutside , because this has direct consequences forth e survival ofth e offspring. Furthermore, Maculinea nausithousi sth e only Maculinea species which can for long periods survive onver y small and dynamic habitats such as road verges andcana l borders (Ebert & Rennwald 1991). Undersuc h conditions,i t seems naturaltha t this relatively mobile parasitic butterfly (Binzenhofer in Settele efal. 1996 ,ow n unpublished data) can detect its host antspecies , and successfully colonise suitable locations, rather than loose many offspring by random oviposition onsink s of Sanguisorba officinalisplant s without Myrmica rubra.

Consequences forsuccess ofreintroduction

After their release,th e butterflies were able to colonise the best locations first andestablis h onth e siteswher e ecological requirements are best realised.Afte r reintroduction, butterfly numbers have increased more than expectedfro m one yeart oth e next. Maculinea nausithouseve n managed to leaveth e reintroduction site and settle on alocatio nwit hMyrmica rubra,tha t wasthough t to beou t of reach ofth e butterflies, because iti s separated from the release site by high poplars,willow s andtal l herb vegetation. Forth e success ofth e reintroduction, it is important to know whether the limited number ofegg s ofth efounde r females will be deposited accordingt oth e randomovipositio n hypothesis oraccordin g toth eant - mediated oviposition hypothesis. Inth e latter case,undesirabl e losses inth e criticalfirs t founding years will beavoide d andth e chance of successful establishment will increase. For thesam e reasons,smal l populations of Maculineanausithous and M.teleius anddispersin g individuals profit from

114 Chapter 5:Habitat selection

beingabl et o select between sources and sinks.Thi s is important for highly endangered animal species witha ver y specialised life cycle asthes e butterflies.

Acknowledgment

We thank Maria Asuncion Hidalgo Lopezfo r collecting the ant samples atth e meadow BW.Andre a Grill, Maarten van Steenis, Gerrit Klompan d Menno vanZuije n helpedwit hth e collection offlowerheads . We areals o indebted to Esther Rebon Sartal andJavie r del Rio Diezwh o dissected so manyflowerhead s and counted the butterfly eggs. Paul Kreijger counted the butterflies according toth e Dutch Butterfly Monitoring Scheme andgav e us valuable records of dispersing Maculineabutterflies .Victo r Mensing helped usa lotwit hth edat a input. MichielWalli sd eVrie s correctedth e Englishan d gave invaluable comments to improve the manuscript. Manythank s alsot o Chris van Swaay, Karle Sykora, Ivo Raemakersan dAndr e Schaffers for inspiring discussions and comments ona nearlie r draft ofthi s manuscript. The Dutch State Forestry Service kindly gave us permission to conduct this study inth e nature reserve Moerputten.Th e contribution of FLwa s financially supported byth e Netherlands Organisation for Scientific Research (NWO) (Stimulation Program Biodiversity "Development of strategies for conservation and restoration of biodiversity inagricultura l areas").

115 Chapter 6: Colonisation and dispersal

Landscapeconstraints inthe dispersalof two parasitic butterfly species

Summary

Inthi s paper, we investigate the population responses oftw o butterfly species, Maculinea teleius and M. nausithous, within a restricted set of patches inwhic h the habitat quality is spatially heterogeneous. This differs from metapopulation studies where the dynamics of local populations andth e exchange of individuals are studied in spatially disjoint habitat patches that are assumed to contain uniformly suitable habitat. The presence of both butterfly species largely depends on the presence of two larval resources: the host plant Sanguisorba officinalis and host ant nests.Althoug h the adult individuals of both butterfly species can select sites that contain these resources, dispersal leading to colonisation of suitable sites at a larger spatial scale seems to be constrained.Th e question iswhethe r the spread of these two butterfly species is limited by the quality or the spatial arrangement of their habitat. We addressed this question by investigating distribution shifts inth e two butterfly species since their reintroduction and establishment inth e nature reserve Moerputten and surroundings inTh e Netherlands. We examined for each species whether the colonisation of unoccupied locations and the abandonment of occupied locations are related to their degree of connectivity. According to the random sample hypothesis, no effects of the degree of connectivity are expected. Effects of the degree of connectivity on colonisation and abandonment suggest that these two processes are not random with respect to the degree of connectivity of the plots.W e show that dispersal constraints at the individual level may affect habitat selection and have an effect on the colonisation and abandonment rates in the populations of the two Maculinea butterfly species andthu s on their distribution.W e discuss the consequences for conservation ofth etw o species after reintroduction.

Van Langevelde F, IWynhoff , HOlff , CAM van Swaay, PM Brakefield & HHT Prins. Submitted.

117 Chapter 6: Colonisation and dispersal

Introduction

Effects ofth e spatial arrangement of habitat on population dynamics haveofte n been investigated inth e context of metapopulations,wher e there are local populations that interactthroug h dispersing individuals. One characteristic of metapopulations isth e dynamic equilibrium between colonisation of patches of habitat andextinctio n of local populations (Verboom etal. 1991, Hanski 1999). Metapopulationstudie s are mainly basedo n local populations inspatiall y disjoint habitat patches that are assumed tocontai n uniformly suitable habitat. Insevera l ways, however, these assumptions violate realworl d situations. For many species, local populations are difficult to distinguish and densities vary over space related to local habitat quality (Harrison 1994a, Lewis etal. 1997 ,Thoma s & Kunin 1999).Withi n and between sites, habitat quality isofte n spatially heterogeneous which mayaffec t habitat selection and movement patterns (Morris 1995, Doak 2000,Gutierre z etal. 1999, Haddad 2000).W e investigate the population responses oftw o butterfly species, Maculinea teleiusan d M.nausithous, within a restricted set of patches inwhic hth e habitat quality isspatiall y heterogeneous. Incontras t to metapopulation studies with local populations inhabita t patches, colonisation is here defined asth e process that empty plotswithi n a habitat patch becomeoccupied . Thetw o butterflies are strictly sedentary, with Maculinea nausithous moving,o naverage , larger distances than M.teleius (Settel e 1998).Th e presence of both butterfly species largely depends onth e presence oftw o larval resources. First, both Maculineateleius an d M.nausithous deposit their eggsonl y onth e host plant Sanguisorba officinalis(Thoma s 1984, Elmes& Thomas 1987,Wynhof f etal. 2001a,chapte r 5). The early instar caterpillars feed ondevelopin g seeds ofth e host plant. Second,bot h species are obligate myrmecophiles sincethei r later instars are hosts inan t nestswher e they feed onan t larvae.Afte r twot othre e weeks,fourt h instar caterpillars leavethei r host plants to befoun d by Myrmica worker antsan d aretake nt o their underground nests.Maculinea teleius mainl y parasitises onth ean t Myrmica scabrinodiswhil e Maculineanausithous ca n befoun d in nests of Myrmicarubra (Thoma s etal. 1989).Wynhof f etal. (2001a ,chapte r 5) analysed the habitat selection ofthes e butterflies, measured byth e oviposition behaviour and the occurrence of adults,an d showedtha t females of both butterflies are ablet o quite reliably select those host plants for oviposition whichar e inth e proximity of nestso fthei r specific ant host. In 1990,th etw o butterfly specieswer e reintroduced inth e Moerputten nature reserve inth e Netherlands following their national extinction in 1976

118 Chapter 6: Colonisation and dispersal

(Wynhoff 1998,chapte r 3).Thei r reintroduction can be rated as successful since both butterfly species have established themselves atsite s where they were released.However , although suitable habitat seemst o beavailable ,th e distribution ofth etw o butterflies is restricted: Maculinea teleiusoccur s only onth e meadowswher e itwa s reintroduced,whil eM. nausithousha s established populations onth e railway embankment andth e surrounding roadverge s (Wynhoff 1998,chapte r 3).Withi n a habitat patch,female s are ablet o preferentially laythei r eggs in plotswher e bothth efoo d plant andth e specific host ant occur (Wynhoff etal. 2001a ,chapte r 5). However, the species does notsee mt o havea highrat eo f colonisation of more distant plotswher e both resources also occur. The question isthe nwhethe r sucha spread ofthes etw o butterfly species islimite d byth equalit y orth e spatial arrangement ofthei r habitat. We addressedthi s question by investigating distribution shifts inth etw o butterfly species since their reintroduction and establishment. By collecting data atth e level of 1x 1m plot s inal l patches with host plants inan d around the nature reserve,w e studied theeffect s of within and between habitat heterogeneity on butterfly behaviour and distribution. Metapopulation theory predicts that the degree of habitat connectivity is an important landscapefeatur e that influences colonisation of unoccupied locations (Fahrig & Merriam 1985, Hanski 1994,Wit h &Chris t 1995, Gutierrez etal. 1999 ,Va n Langevelde 2000). Inthi s context, connectivity is thedegre et owhic hth e landscapefacilitate s or impedes movement among resource patches (Taylor etal. 1993) . Forth e butterfly species, connectivity will beexpresse d asth e nearest neighbour distance between plots. In conditions of low connectivity, dispersal may beconstraine d resulting ina lo w probability ofcolonisatio n of unoccupied locations.Accordin g toth e random sample hypothesis (Connor & McCoy 1979, Hailaet al. 1993 ,Andre n 1996, 1999), however, the simplest explanation for alo w colonisation probability of unoccupied locations isthei r low habitat quality. Then,th e colonisation pattern of locationswit ha lowdegre e ofconnectivit y isa random sample from equally large locations incontiguou s habitat ofth e same quality. In particular, the random sample hypothesis predicts noeffec t ofth edegre e of connectivity ondistributio n and colonisation. Forth etw o butterfly species,w e testwhethe r the colonisation of unoccupied locations is relatedt othei r degree of connectivity. Besidesth e quality ofth e habitat,th e risktha t alocatio n becomes unoccupied isassume d todepen d onth e migrationfro m other occupied locations, i.e.,th e rescue effect (Brown & Kodric-Brown 1977).Th e rescue effect refers to increasing the size of local populations and hence decreasing

119 Chapter 6: Colonisation and dispersal

their risk ofextinctio nwit h increasing rate of immigration. Itimplie s a positive relationship between thefractio n of occupied locations andth eaverag e size of local populations or densities. Thus,a negative relationship canb e expected between the migration rate of alocatio n andth e probability that this location becomes unoccupied (Hanski 1999,Stace y etal. 1997).Whe nth e spatial arrangement ofth e habitat has noeffec t onth e population responses, this rescue effect does not depend onth e degree of connectivity ofth e locations. Forth etw o butterfly species,w e test whether the abandonment of occupied locations is relatedt othei r degree of connectivity.

Materialand Methods

Studyarea The study area covers the nature reserve of Moerputten(11 5 ha) and itssurroundings , situated inth ecentr e of the Netherlands inth e province of Noord-Brabant (51°41'N,5°5'E ,altitud e 2 mabov e sea level,figur e 6.1, Wynhoff 1998,se e chapter 1 for further information). The area consists ofa lake, surrounded bytal l swamp vegetation andwillo w and elder forests, and several moist meadows atth e higher partstha t contain the habitat ofth e butterflies. In 1990,th efoundin g butterflies were released onth e meadows at the southern border ofth e reserve (figure 6.1). Maculinea teleiuscolonise d the release sites andexpande d to afe w meadowsfurthe rwest .Afte r threeyears , however, this specieswa s restricted only toth e release site,wher e itstil l occurs today. Maculinea nausithous apparently did not establish itself inth e nature reserve after release, butwa sfoun d ayea r later onth e railway embankment crossing the reserve,S Di nfigur e 6.1. The latter population rapidly increased and gave rise toth e colonisation of habitat at adistanc e of about 500 mo nth e verges ofth e minor road RD(figur e 6.1). Whileth e railway embankment population was stablefo r several years andthe n decreased innumbers ,th e RD- population increased rapidly.Anothe r local population wasfoun d 2year s later at adistanc e of 5km .Thi sthir d population,occupyin g roadverges , is still very small.Thi s unexpected large-scale colonisation is not included in this study sincew efocu so nth e localcolonisation s within the nature reserve anddirec t surroundings.

Butterflydata Both Maculinea teleiusan d M.nausithous hav e been continuously monitored sinceth e reintroduction. Butterflies were counted by meanso f

120 Chapter 6: Colonisation and dispersal

transect counts and mark- recapture studies (Wynhoff 1998,Wynhof f etal. 2001a, 2001b, chapters 3, 5an d 7). Furthermore,observation s of butterflies duringfiel d visits were recorded.Th e area of Moerputten and surroundings were visited at least once aweek , and preferably every second orthir dda y during thewhol efligh t period of bothspecies . Inth e study area,58 7 plots sized 1* 1m wer e randomly locatedo n meadows,verge s and patcheswit h Sanguisorba officinalis. Onth e release meadow, BW,w e used a 10 * 10m gri d square system,wherea s the plots were located randomly inth e other patches.Th e size ofth e plots represents the scale atwhic h afemal e of each species decides whether to deposit an eggo r not (Wynhoff efal. 2001a , chapter 5). Ineac h plot,th e presence or absence of anadul t butterfly was recorded during the 10year s following reintroduction.A colonisation was recorded when a plotwa s unoccupied ina certain year andoccupie d inth e next year. The opposite,abandonmen t ofa plot,wa s recorded when aplo twa s occupied ina certai n year and unoccupied inth e nextyear . For Maculinea teleius,al lyear s of observations were used inthi s study. Forth e colonisation analysis,th e year of reintroduction was excluded since the butterflies could not spontaneously colonise thefirst-yea r occupied plots. Inaddition , Maculinea nausithous passedthroug h a bottleneck of small numbers in 1991and ,therefore ,th e analysis begunwit hth e census in 1992. The degree of connectivity of aplo twa s measured asth e Euclidean distance toth e nearest-neighbour plot thatwa s occupied (from centret o centre). Todetermin e the relationship between the probability of both colonisation andabandonmen t ofa ploti nyea r fan d itsdegre e of connectivity, we usedth edistanc e to its nearest neighbour thatwa s occupied inyea r f -1.

Vegetation data Since Maculineabutterflie s only occur at siteswit hthei r host plant,w e restricted theanalyse s tothos e plotswit h Sanguisorba officinalis(figur e 6.1). For asubse t of25 1 plots,dat a onvegetatio n composition were collected through vegetation releves according toth e Braun-Blanquet method. Inthes e plots,th e maximum height ofth evegetatio nwa s measured (incm) . Thevegetatio n composition was analysed using a Detrended Correspondence Analysis (DCA,Jongma n era/. 1995),i nwhic h samples and species areordinate d alongcanonica l axes according to similarities in occurrence.A sa measurement for vegetation composition,th e sample scores of the plotsfo r thefirs t 4 axeswer e takenfo r further analysis.

121 Chapter 6: Colonisation and dispersal

-,. Mj.ffl »ft /

-TV

"* ,*^-^-*Kl^ JT"~~ \ s *v \j / ••> '

Figure 6.1: Study area of Moerputten and its surroundings inth e centre of the Netherlands. Indicated with 'n'an d 't' are the meadows were the two butterfly species, Maculinea nausithous and M.teleius respectively, were released in 1990. The host plant Sanguisorba officinalis occurs inth e grey areas. Dots denote single plants or groups of plants. BW,W H and PN areth e meadows, RD and MPP are the roadverge s and SD is the railroad embankment.

Furthermore,w e calculated the mean Ellenbergvalue s for moisture, productivity and acidityfo r each plot (Ellenberg 1992). For details ofth e analysis seeWynhof f et al. (2001a,chapte r 5).

Antdata The composition ofth e localan tfaun a andth e density of host ant nests depend onth evegetatio n structure and local microclimate (Elmes etal. 1998,Wynhof f et al. 2001a). Per plot,th e presence of ant species was examined byattractin g themwit h sugar cubes.Th e sugar cube represents the caterpillar waiting to befoun d byworke r ants. Inth e centre ofth e plota t thefoo t of a Sanguisorba officinalisplant , asuga r cubewa s placed ona concave plate covered with black plastic. Thiswa s done inth e early morning hours beforeth e ants start their daily activity period (before 8a.m.) . After at least 1 hour, the baitswer e checked forworke r ants visiting the sugar and the ant species were identified. Inth e evening,al l baitswer e removed.Sinc e

122 Chapter 6: Colonisation and dispersal

Myrmica ants have arestricte d foraging range of 2 maroun d their nestsit e (Elmes etal. 1998) ,w e assume that we onlyfoun d workers from nests inth e close proximity ofth e Sanguisorba plant during the short recording period. Moreover, weassum e thatth e plotswher e sugar cubes remained undetected represented ant-free plots.Sinc eth e number ofworke r antso n the bait represents neither the density of ant nests north e size ofth e nests in the plot,w e only used the presence orabsenc e of anan tspecies .

Statistical analysis Regardingth e abundant host plant Sanguisorba officinalisan dth e low nestdensit y ofth e host ants,th e quality ofth e habitat of both species is largely determined byth e presence ofth e ant nests. Inthei r habitat selection study,Wynhof f etal. (2001a ,chapte r 5)foun d that the probability that an adult butterfly was present ina plot , is relatedt oth e presence of their Myrmicahos t ant,th evegetatio n height andvegetatio n composition as represented byth efirs t DCA-axis (Table 6.1). Based onthes e results,w e describe the quality ofth e habitat for eacho f the butterfly species asfollows :

+ 2 Qtei = Po PiMs +P 2 °CA +P 3 DCA +p 4VH for M. teleius(eq .1 a )

+ Qnaus =Y o YiM r+ y 2 °CA +y 3VH for M. nausithous(eq . 1b )

where p: andy-, areth e regression coefficients (Table 6.1), Msan d Mrth e host ant presence of respectively Myrmicascabrinodis and M.rubra, DC Ath efirs t DCA-axis andV Hth evegetatio n height. Thefirs t DCA-axis correlates with the Ellenberg productivity (n =234 , r= 0.887 , p< 0.001) , acidity (n= 229 ,r = 0.723, p< 0.001 )an d moisture (n =236 , r= -0.626 , p< 0.001 ) (Wynhoff et al.2001a ,chapte r 5). Thevegetatio n releves areordinate d from the most nutrient poor meadows (BW,WH) ,vi a richer meadows and roadverge s (RD, MPP)t oth etal l herbvegetatio n atth e railroad embankments (SD) (Figure 6.2).

The colonisation of unoccupied plotsan dth eabandonmen t of occupied plots byth etw o butterfly species were analysed byfittin g a logistic regression.Sinc eth e probability that aplo to f certain quality iscolonise d or abandoned inyea r fdepends ,amon g others,o nth e population size intha t year, we usedth etota l number ofoccupie d plots as explanatory variable as well. Inth e regression analyses of both colonisation and abandonment, first the habitat quality andth e total number ofoccupie d plotswa s added.Then , the distance toth e nearest-neighbouring occupied plotwa s added. Duet o

123 Chapter 6: Colonisation and dispersal

the constant management ofth e nature reserve andth esurroundin g habitat, we consider thevegetatio n composition andstructur e andth e presence of ant nests constant over allyear s inth e analyses.

Results

Inth estud y area,27 4 colonisations of atota l of 1920unoccupie d plots were observed for Maculinea teleius, while 256 of atota l of 590 occupied plotswer e abandoned the nextyear . Maculinea nausithous colonised 50o f 1849unoccupie d plots. From 144occupie d plots,5 1wer e abandoned the

Table6.1: Results of the logistic regression analyses of the presence/absence of Maculinea teleius and M. nausithous inth e study area (Wynhoff efal. 2001a) . The regression coefficients Pi and Y,ar e given and used as in eq. 1aan d 1b. DCA represents the first DCA-axis of the

vegetation releves, Ms and MRi s the presence/absence of a host ant nest (Myrmica scabrinodis and M. rubra respectively) andV H is the vegetation height incm . The probability of observing a butterfly of either one or the other Maculinea species (M) is given by: ln[M/(1-M)] =constan t +Z regression coefficient x variable.

Maculinea teleius M. nausithous number of cases 1900 1710 % predicted correct 76 94

Maculinea teleius variable P S.E. Wald Sig. Constant -3.750 0.395 90.092 0.000 Population size 0.019 0.002 76.678 0.000 DCA 4.232 0.687 37.984 0.000 DCA2 -3.082 0.422 53.420 0.000

Ms 0.439 0.131 11.313 0.001 VH 0.027 0.011 5.788 0.016

Maculinea nausithous variable y S.E. Wald Sig. Constant -7.505 0.522 206.964 0.000 Population size 0.018 0.005 14.692 0.000 DCA 1.766 0.156 128.032 0.000

MR 1.147 0.230 24.919 0.000 VH -0.009 0.004 4.189 0.041

124 Chapter 6: Colonisation and dispersal

following year.Th etota l population sizewidel yfluctuate d between sequential years:fo r Maculineateleius, the average number of occupied plots is75. 3 witha standar d deviation of 29.9 (n= 9) ,whil e M.nausithous hasa n average of41. 3 occupied plotswit h astandar d deviation of 21.2 (n= 7) .

The probability that anunoccupie d plot iscolonise d is significantly relatedt oth e recorded habitat quality ofth e plot,th e overall population size and its degree of connectivity (Table 6.2). For each butterfly species,a similar seto f variables yielded significant effects onth e colonisation probability. Increasing habitat quality leadst oa n increase in colonisation probability. However, plotswit h habitat of high quality can remain unoccupied whenthe y have a low degree of connectivity since wefoun d adecreas e in colonisation probability with increasing distance toth e nearest-neighbouring occupied plot. Plots of low habitat quality and highdegre e of connectivity may becolonise d morefrequentl y thanexpecte d basedo nth e random sample hypothesis. Figure 6.3 gives the predictions forth e colonisation probability of unoccupied plots byeac h butterfly species.Thi sfigur e shows the distances that individuals of each species covered to colonise unoccupied plots: Maculinea teleius,o naverage , showed shorter distances

(M 3,0 " (A X • < 3' O BW- ^^ O ~y ^\ MPP 2,5- / WH X ^ .«, /- -^ / s^~ / —"-s^. ^~x\ / /X. ^"x* / PN( J.°**°X \ i* . \. \

• \ • •*• 4° i° «T\ \ ft y^ r\ \ A * \ 7 ^ • V %*<. • °\ * \\ / \ \ \ \ * • •N& x •* o J \/. • \ \ v * \SD T •. .'.•V*:X^X^ / Y \ °\ °\ * * \ I \ » \ p « A\ . \ • •» / V" Px? J / •.*••• / \ ^X °"1 ^X y 0,5 - V* .• • / •* V * * 7— X . * • * / RD\ /

c) 0,5 1 1,5 2 2,5 3 3,5 4 4 5 DCAaxi s1

Figure 6.2:Vegetatio n releves from the study area along the first two canonical axes. The meadows are BW, WH and PN,th e road verges are RD and MPP,th e railroad embankment is SD (seefigur e 6.1).

125 Chapter 6: Colonisation anddispersal

Table 6.2: Results ofth e logistic regression analyses ofth e colonisation of unoccupied plots by Maculinea teleius and M.nausithous inth estud y area. QteH andQ nausrepresen tth e habitat quality forMaculinea teleius and M.nausithous, respectively (seeeq . 1aan d1b) . The distance refers toth edistanc e toth eneares t neighbour plottha t wasoccupie d in the previous year.

Maculinea teleius M. nausithous number of cases 1920 1849 % predicted correct 87 97

Maculinea teleius variable regr. coef. S.E. Wald Sig. Constant -1.249 0.502 6.192 0.013

Habitat qualityQ tel 0.693 0.138 25.101 0.000 Population size 0.025 0.003 74.818 0.000 Ln(Distance) -1.092 0.126 75.181 0.000

Maculinea nausithous variable regr. coef. S.E. Wald Sig. Constant -6.995 1.124 38.730 0.000

Habitat quality Qnaus 0.604 0.082 54.515 0.000 Population size 0.015 0.007 5.543 0.019 Ln(Distance) -0.422 0.143 8.736 0.003

than M.nausithous. Colonisation distances arehighl y skewed,especiall yfo r Maculineateleius. This species hasa clumpe d distribution inth estud y area, resulting inman y examples oflarge r distances toapparentl y suitable,bu t unoccupied plots (Table 6.4).Th ehighes t meanan dmaximu m colonisation distances were measured inyear sfollowin g population increase, e.g.,i n 1991afte r reintroduction.Althoug h ahig h population size increasesth e probability ofcolonisation ,w ecoul d not find evidence that dispersal distances aredensity-dependent : individuals dono tcove r larger distancest o colonise unoccupied plotswhe nth epopulatio n size ishigh .W e tested this addingth einteractio n betweenth edegre e ofconnectivit y andth eoveral l population sizet oth eregressio n modelfo rMaculinea teleius, bu ti twa sno t significant. Maculinea nausithousha s colonised plotsa tlarge r distances compared to M.teleius and hasestablishe d twoloca l populations. Therefore, the distances tounoccupie d plots areshorte r (Table 6.4).Again ,n osignifican t density-dependent colonisation distances werefound .

126 Chapter 6: Colonisation anddispersal

„„

OS- Maculineateleius

0.7 • fi*> It*'- cte d probabilit y £ a- 0.3-

0.2-

0.1 • • • }tit' ' ' • ..T". " . •••fit •".'•". , •j.'...Kj^.-^j-ad.i^A:t^kL>jL«: 0 20 40 60 SO 100 120 140 160 180 200 distance (m) Maculinea nausithous

0.5-

0.4-

0

O 0.3- ••"o Q. o T> ?. • $<>•'' » o

? 0.2- #.•••• •' • •*

0.1 •

0 50 100 150 200 250 300 350 400 distcrice(m) Figure 6.3:Prediction s ofth e colonisation probability plotted againstth e observed distances ofeac h unoccupied plot to its nearest-neighbouring plot that was occupied thepreviou s year. Open squares denote colonised plots, dots refer to plots that remained empty.

127 Chapter 6: Colonisation and dispersal

As can beexpected ,th e probability that anoccupie d plot is abandoned increases for both butterfly species whenth equalit y ofth e habitat is lower. The probability of abandonment in Maculinea nausithous could only be relatedt o habitat quality (Table 6.3). For Maculineateleius, habita t quality of the plot,th e overall population size and itsdegre e of connectivity affect the abandonment probability. Whenth e distance toth e nearest neighbouring occupied plot increases,th e probability that a plot isabandone d by Maculineateleius increases ,to o (Table 6.3).

Discussion

The results show that bothth e quality andth e spatial arrangement of their habitat limit the spread of thetw o Maculineabutterfl y species.W e can distinguish two scale levels atwhic h these factors operate (Haila efal. 1993 , Morris 1995):a tth e level ofth eadul t individual,th e spatial habitat arrangement may affect the selection of oviposition sites,wherea s atth e level of populations theexchang e of individuals among local populations is affected. Wefoun dtha t although individuals can select their habitat, low quality plots are sometimes occupied and high quality plots can remain unoccupied especially whenthei r degree of connectivity is low.Thi s constrained dispersal has aneffec t onth e rates of colonisation and abandonment, andthu s onth e local distribution ofth e species. Constraints in dispersal setth e conditions for the selection of habitat. This phenomenon belongs toa centra l issue inecology , namely the problem of scale: patterns at one scale may be manifested at another (Wiens efal. 1993 , Levin& Pacala 1997).

Thetw o butterfly species are highly specialised since they require host plants and host ant nestsa s larval resources.Thi s specialisation appears to causethei r restricted spatial distribution inth e study area. Low quality ofth e habitat, interm s ofth e lacko f one ofth e two larval resources, maythe n limit the spreado f the butterflies whenthe y occur insmal l local populations,suc h as after a reintroduction or acatastrophi c event. Sinceth e host plant Sanguisorba officinalisi sabundan t whilstther e isa lo w nest density ofth e host ants,th equalit y ofth e habitat of both butterfly species is largely determined byth e hostant . Nevertheless, both Maculineateleius an dM. nausithous are able to selectfo r Sanguisorba officinalisi nth e proximity of host ant nestst odeposi t their eggs (Wynhoff efal. 2001a , chapter 5). For Maculineateleius, however , Wynhoff efal. (2001c ,chapte r 4)foun da

128 Chapter 6: Colonisation and dispersal

Table 6.3: Results of the logistic regression analyses of the abandonment of occupied plots

by Maculinea teleius and M. nausithous in the study area. Qte,an d Qnaus represent the habitat quality for Maculinea teleius and M. nausithous, respectively (see eq. 1a an d 1b) . The distance refers to the distance to the nearest neighbour plottha t was occupied in the previous year.

Maculinea teleius M. nausithous number of cases 590 144 % predicted correct 68 72

Maculinea teleius variable regr. coef. S.E. Wald Sig. Constant -1.411 0.848 2.770 0.096

Habitat quality Qle, -0.619 0.210 8.679 0.003 Population size -0.026 0.004 48.658 0.000 Ln(Distance) 1.976 0.337 34.483 0.000

Maculinea nausithous variable regr. coef. S.E. Wald Sig. Constant 5.788 1.512 14.661 0.000

Habitat quality Qnaus -0.648 0.152 18.247 0.000

positive relationship between ant nest presence and oviposition atth e beginning of thefligh t period,bu t this relationship disappeared atth e endo f thefligh t period.Moreover , Wynhoff etal. (2001a , chapter 5)foun d that Maculinea nausithousdeposite d 26%o fth e eggsdurin g thewhol e flight period at plotswithou t ant nests.Althoug h there are differences in oviposition behaviour betweenth etw o butterfly species (Thomas 1984, Figurny& Woyciechowski 1998),thes efinding s strongly suggesttha t there are limitations onth e selection of oviposition siteswhe nth e majority of suitable flowerheads inth e proximity of butterflies have already been ovipositedon . Based on our results,th e observed limitations in habitat selection canb e accountedfo r byconstraine d dispersal.

Connectivityand colonisation

For both Maculineanausithous and M.teleius, w eteste d whether plots further awayfro m occupied plots have alowe r probability of beingoccupied . The random sample hypothesis provided the null hypothesis:ther e isn o effect ofth e degree of connectivity onth e probability that an unoccupied plot

129 Chapter 6: Colonisation and dispersal

iscolonised .Th e results showthat , onth e one hand,plot so f low habitat quality and high degree of connectivity may becolonise d morefrequentl y than expected basedo nth e random sample hypothesis. Onth e otherhand , plotswit h high habitat quality but lowdegre e of connectivity are less frequently colonisedtha nexpected .Thu s colonisation isno t randomwit h respect toth edegre e of connectivity ofth e plots.

The random sample hypothesis has been previously examined for metapopulations of other species, e.g.,b yVerboo m etal. (1991 ) andAndre n (1996, 1999).Thes e studies, however, focused onth e question ofwhethe r the distribution of aspecie s ina heterogeneous landscape issolel y duet o habitat loss or isals o duet o habitat fragmentation. Here,w e discuss another dichotomy, namely whether the distribution of aspecie s ina heterogeneous landscape isdu et odifference s in habitat quality or also duet o habitat fragmentation. Moreover in manystudie s of spatial population ecology, local populations can bedistinguishe d anda constan t habitat quality ofth e patches isassumed . Inou r study,th e seto f habitat patches,th e meadows,i s actually aspatiall y heterogeneous mosaic of plotswit hdifference s in vegetation structure and ant nest presence.

Table 6.4:Som e characteristics of the degree of connectivity of plots that remain unoccupied and are colonised by Maculinea teleius and M. nausithous in and around the study area. The degree of connectivity is expressed in the distance (in m)t o the nearest neighbour plot that was occupied in the previous year. The skewness is a measure of the asymmetry of a distribution: a large positive skewness (more than twice its standard error

SESK) indicates a long right tail. This means that the majority have dispersed short distances (seefigur e 6.3).

n Median Minimum Maximum Skew­ SESK plots distance distance distance ness

M. nausithous All 1849 143 5 655 0.620 0.06 Empty 1799 147 5 655 0.061 0.06 Colonised 50 42 6 442 1.560 0.34

M.teleius All 1920 81 5 922 1.190 0.06 Empty 1646 171 5 922 1.011 0.06 Colonised 274 9.8 5 263 7.900 0.30

130 Chapter 6: Colonisation and dispersal

Thecolonisatio n events of Maculinea teleiusmainl y took placewithi n the meadowwher e the butterflies have occurred sincethei r release in 1990. The very small colonisation steps (Figure 6.3,Tabl e 6.4) indicate an expansion- retraction process. This is most likely caused by stochastic demography duet oweathe r conditions.Th e probability that an individualwil l leavethi s meadow islow .Thi s agreeswit hth e displacement of individuals in a mark-recapture study. Eventhoug h the butterflies arequit e active,the y tendt o stay at particular siteswithi n the meadows, resulting ina mean displacement of lesstha n 1m pe rda y (ina 10* 10m grid) .Th e maximum displacement found was 260 mi n 10day sfo r males and 639 mi n7 day s for females (unpublished datafro m 1991).Simila r data have beenfoun d inothe r populations (Settele 1998),wit h asingl e displacement of moretha n 5km . Dispersal to unoccupied,suitabl e habitat plots at longer distances seemst o beconstraine d andthi s islikel y to limitth e distribution of Maculinea teleius. Thefe w long distance movements may bever y important for the persistence ofth e population asthe y enable exchange between local populations (cf. Quinn & Hastings 1988).

Maculinea nausithousha s beenfoun dt o colonise unoccupied plots less frequently, but it covers,o naverage , longer distances.Th e latter isshow n by the longer colonisation distances (Figure 6.3,Tabl e 6.4) and by mark- recapture studies. Meandail y displacement was 6 mfo r females and 8 mfo r males (unpublished datafro m 1991) and displacements exceeding 1k m haveonl y rarely beenfound ,bu t morefrequen t than inM. teleius (Settel e 1998). However, with a maximum colonisation distance of44 2 m,w e conclude that the spatial arrangement ofth e habitat limits the distribution of Maculinea nausithous.

Connectivityand abandonment

The rescue effect reduces the risk of extinction orabandonmen t by migration from other occupied plots.Th e results in Maculineateleius sugges t the existence of such a rescue effect: the probability of abandonment ofa n occupied plotdecrease s with both overall population size andth edegre eo f connectivity ofth e plot.Wit h increasing overall population size (more occupied plots),mor e individuals tendt o move toa particular plotwhic h leadst o an increase in its local population density and hence adecreas e in the risk of abandonment ofthi s plot.

131 Chapter 6: Colonisation and dispersal

Females partly selectflowerhead s without ant nests inth e proximity, especially atth een d ofth efligh t period (Van Dyck etal. 2000 ,Wynhof f etal. 2001c, chapter 4). They select for empty flowerheads to oviposit, butthe y are restrained tocove r large distances tosearc h for theseflowerheads . Inthos e plotswithou t ant nests,n ocaterpillar s pupate andabandonmen t will be recorded unless the searching females again oviposit their eggso nth e flowerheads. Partly,thi s may appear as a rescue effect although infac t source-sink relationships appear. This may be more obvious in Maculinea teleiusthe n in M.nausithous since the latter canoviposi t several eggso nth e sameflowerhead . Hanski etal. (1995 ) showth eexistenc e ofth e rescue effect ina metapopulationo fth e butterfly Melitaeacinxia. They coulddistinguis h this effect for different population sizes,findin g a reduction inloca l extinction in small populations and not in large populations.Th e rescue effect was also demonstrated in patchy populations ofAphantopus hyperantus(Sutcliff e et al. 1997).

Implications forconservation

Although individuals of Maculinea teleiusan d M.nausithous are ablet o select plotswit hth etw o larval resources, dispersal isconstraine d duet oth e spatial arrangement ofth e habitat. This limits the spread ofsmal l populations, inou r casefollowin g reintroduction ofth e species.Althoug h the population dynamics of both butterfly species areaffected ,w e expect that Maculineateleius i sles swidel y distributed than M.nausithous sinc e they coversmalle r distances (Figure 6.3). Thisagree swit hth e observed distribution pattern overth e years. Moreover, we predicttha t the population of Maculinea teleiuscoul d be more stable than M.nausithous sinc ew e found, onaverage ,a lower extinction risk duet oth e rescue effect inM. teleius.Thi s also agreeswit hth eobservation s todat e since thevariatio n in overall population size is higher in Maculinea nausithoustha n inM. teleius. Conservation of Maculinea teleius should concentrate on improving local habitat quality,a sth e persistence of this species isdependen t onth e situation ofth e core population.Th e constrained dispersal inthi s butterfly species resembles almostflightles s moth Itameandersoni (Doa k 2000) and theflightles s moth Orgyiavetusta (Harriso n 1994b). Giventh e low dispersal abilities,expansio n ofth e resident population can only beexpecte d on high quality patches inth e close proximity. Plotswit h high quality habitat at longer distance are more likely to becolonise d if highqualit y stepping stones are available in between (Haddad2000) . Conservation of Maculinea nausithous

132 Chapter 6: Colonisation and dispersal

bycreatin g aspatia l network of suitable habitat plotsseem s to be more effective since this species isabl et o cover longer distances. Local populations ofthi s species are generally smaller and depend moreo n exchange of individuals.

Studies inspatia l population ecology focus oneffect s ofth e degreeo f connectivity while habitat quality issubjec t in habitat selection studies.A n explicit link between thetw o isofte n lacking (Lima &Zollne r 1996). Inou r study,w e show that dispersal constraints atth e individual level may affect habitat selection and have aneffec t onth e colonisation and abandonment rates inth e populations ofth etw o Maculinea butterfly species andthu s on their distribution. Understanding the scaling ofth e processes isrelevan tfo r conservation since itcontribute s to insight inth e bottlenecks inth e distribution of species.Therefore , such links between processes at individual and population levelshoul d bea n urgenttopi c inconservatio n biology.

Acknowledgements

Wear ever y gratefult o Paul Kreijgerwh o countedth e butterflies accordingt oth e Dutch Butterfly Monitoring Scheme and gave usvaluabl e records of dispersing Maculineabutterflie s and potential habitats. Maria Asuncion Hidalgo Lopez collected the ant samples atth e meadow BW. Marcel Grutters,Andre a Grill, Maarten van Steenis, Gerrit Klompan d Menno vanZuije n helpedwit h thevegetatio n measurements andth e other ant samples.Victo r Mensing helped usa lotwit hth e data input.Th e Dutch State Forestry Service kindly gave us permissiont oconduc tthi s study inth e nature reserve Moerputten.Th e contribution of FLwa sfinanciall y supported byth e Netherlands Organisation for Scientific Research (NWO) (Stimulation Program Biodiversity "Development ofstrategie s for conservation and restoration of biodiversity inagricultura l areas").

133 Chapter 7:Genetic variation

Genetic variation isretained after reintroduction ofthe butterflies Maculinea nausithous and Maculinea teleius

Summary

Reintroduction is an increasingly important tool in nature conservation after populations of rare animals have disappeared from nature reserves. The demography of the founder populations is often well studied, but there are only afe w case studies of the genetic consequences of the potential bottleneck. The rare butterfly species, Maculinea nausithous and M. teleius, both obligate ant parasites, were reintroduced to a Dutch nature reserve in 1990 using butterflies collected in Poland.Th e population increase was monitored and five years after reintroduction a genetic analysis was performed. The reintroduced populations of both butterfly species did not suffer an initial bottleneck interm s of a very low number of founder individuals. Population numbers increased rapidly and loss of genetic variation was only mild. Low levels of electrophoretic variation of the source populations did not appear to have a negative impact on the establishment from the founder populations. There were shifts inth e flight periods after release and the mean minimal life time of the adults decreased. In both Maculinea species significant differentiation atth e Aco-K locus between source andfounde r population was found.Thes e observations suggest that both species experienced selection duet o the changed ecological conditions in The Netherlands.

Wynhoff I, H deVos , MP van Zuijen, HHT Prins & PM Brakefield 2001. Submitted.

135 •• — Chapter 7: Genetic variation

Introduction

Many populations ofthreatene d animals have disappeared duet oth e destruction ofthei r habitat ordu et o major habitat changes resulting in significantly lower survival probabilities for the species concerned (Hunter 1996,Settel e efal. 1996 ,Soul e 1986). Ifhabitat s are not completely destroyed,conservatio n biologists maywis ht o reconstruct theman d reintroduce lostspecie s (Fischer &Lindenmaye r2000) .Ther e have been many suchfaun a restoration projects,fo r examplewit h numerous butterfly species (Oates &Warre n 1990),th e lynx (Breitenmoser & Haller 1993)an d the beaver (Nolet & Baveco 1996).Althoug h reintroduction maygiv e the appearance of areadil y available tool in nature conservation,succes s isno t guaranteed. Infact , most reintroduction projects in butterflies have been failures (Oates &Warre n 1990).Sometime s reintroductions failed because habitat qualitywa s not good enoughfo r population persistence. Inothe r cases no reasoncoul d befound .Usuall y only the demography ofth efounde r population has been monitored and notth egeneti c consequences ofth e founding event,eve nthoug h they can have astron g impact onsuccess . For thefounder s a reintroduction can becompare d to abottleneck : duet oth e severely reduced population sizether e is likely to bea decreas e of genetic variation and losso f alleles compared toth eorigina l population (Haiti &Clar k 1989, MaynardSmit h 1998).Thi s has beendemonstrate d in bottleneck- experiments with butterflies and other invertebrates under laboratory conditions (McCommas& Bryant 1990,Saccher i etal. 1999) . Reintroduced populations arefrequentl y fully isolated and remain small,s otha t their geneticvariatio n iscompletel y dependent onth e effective founder individuals. Founder populations are comparable toth e remnants of populationfragmentation ,thei r behaviour follows the same rules (Gilpin 1991, Olivieri & Gouyon 1997). Loss ofgeneti c variation can lead to reduced fitness, especially under changing environmental conditions (Hunter 1996). The impact of isolation as aconsequenc e of meta-populationdegradatio n on population genetic structure has been studied inman y butterfly species (Goulson 1993,Haa g era/. 1993,Britte n efal. 1995 ,Johannese n efal. 1996 , Johannesen efal. 1997 ,Meglec z etal. 1997) . Incontrast ,w e know much lessabou tth eeffect s of bottlenecks under natural conditions (butse e Brookes era/. 1997,Barascu d etal. 1999).

In 1990th e Europe-wide endangered butterfly species Maculinea teleiusan d M.nausithous were reintroduced inth e nature reserve "Moerputten" near the cityo f Den Bosch inth e province of Northern Brabant (The Netherlands) (Wynhoff 1998a).Th e last indigenous populations inTh e

136 Chapter 7: Genetic variation

Netherlands were lost inth e late 1970s (Tax 1989).Th efounde r individuals ofth e reintroduced populations of both specieswer e collected from large meta-populations ina strea m valley in Poland.Afte r the reintroduction the populations of bothspecie s developed ina differen t way.Th e density of Maculineateleius immediatel y increased atth e reintroduction site.Th eflight - periodo fthi s species shifted towards afe wweek s earlier overth efirs t three years. Maculinea nausithous onth e other hand,appeare d toexperienc ea second bottleneck. Itdidn' testablis h atth e release site butrathe r elsewhere inth e nature reserve at adistanc e of upt o 200 mfro m the release site. Itha s formed asmal l metapopulationconsistin g oftw o subpopulations. Furthermore,ther e has been only asligh t change inth e flight-period compared toth esourc e population (Wynhoff 1998a). Both species have experienced areductio n in mean minimal lifetim e relative tothos e typicalo f the source population. Herew e report onth e useo f gel electrophoresis to compare source populations and introduced populations for genetic variation after five years of separation.W e investigated whether the reintroduction has involved atru e bottleneck event inth efounde r populations, leadingt o significant losso f genetic variation.W e compare the results from genetic analysis withth e known dynamics of adult populations after reintroduction.

Materials andmethods

Ecologyof Maculinea teleius andM . nausithous Incontras t toothe r butterfly species in Europe,th e larvae of Maculinea species areobligat e parasiteso fants . Maculinea nausithousan d Maculinea teleius oviposit onflowerhead s of Sanguisorba officinalis(Thoma s 1984, Thomas etal. 1989 , Elmes& Thoma s 1987,Wynhof f 1998).Th e early instar caterpillars feed briefly ondevelopin g seeds.Afte r twot othre e weeks,fourt h instar caterpillars leavethei r host plantt o befoun d byMyrmica worker ants andtake n tothei r underground nests.Th e caterpillars feed mainly onan t larvae. The main host antspecie s for Maculinea nausithousi s Myrmica rubra,wherea s Maculineateleius mainl y lives in nests ofMyrmica scabrinodis(Thoma s etal. 1989 ,Wynhof f efal. 2001 , chapter 5).Th e details ofth e life history ofthes e species result inhighl y specialised habitat requirements (Wynhoff etal. 2001, chapter 5, Clarke efal. 1997 ; Figurny& Woyciechowski 1998;Thoma s & Elmes 1998,Thoma s &Wardla w 1992).

The studypopulations The source populations for the reintroduction of Maculinea teleius and M.nausithous are large meta-populations inth evalle y ofth e riverWisl ai n

137 Chapter 7: Genetic variation thevicinit y ofth e village Kostrza near Krakow (Poland) (50°15'N, 19°51'E, altitude 200m above sea level,figur e 7.1). Further information ofthes e populations isgive n by Skalski (1995),Woyciechowsk i (1991) and Figurny& Woyciechowski (1998).Th e size ofth etota l meta-populationo feac ho fthes e species isapproximatel y between 5,000an d 10,000 individuals per year. Although these populations have been decliningfo r many years,w e can assume that they have not undergone any substantial loss of genetic variation through any recent bottleneck.

n^\/

Figure 7.1:Ma p indicating the location of the source (Kostrza) and the founder (Moerputten) populations of the reintroduction of Maculinea teleius and M. nausithous. Abbreviations for countries: PL=Poland, D=Germany, NL=The Netherlands.

In 1990a random sample of Maculinea teleiusan d Maculinea nausithouswa stake n for reintroduction intoth e nature reserve Moerputten in The Netherlands (51°41'N, 5°15'E, altitude 2 mabov e sea level,fo r further description see Wynhoff 1998a). On 30Jul y 1990,3 3 males and 53 (mated) females ofM. teleiusan d 22 males and4 8 (mated) females ofM. nausithous were released. Females probably only mateonc e as onlyfreshl y eclosed females are everfoun d incopula .Th e reintroduced populations were studied ineac hyear .Annua l population sizeswer e estimated by meanso f mark- release-recapture techniques or bywalkin g monitoring transects (Wynhoff 1998a). ForM. nausithous the reintroduction bottleneck has endured for two

138 Chapter 7: Genetic variation

generations, butsinc e 1991th e new population has increased andextende d itsfligh t area.Th e population ofM. teleiusimmediatel y increased, butth e butterflies remained confined toth e single meadow where the introduction was made. In 1995a secon d bottleneck occured duet o accidental mowing of almostth ecomplet e habitat inth e beginning ofAugust .Th e majority ofth e eggsan dth e young larval instars onth eflowerhead s were killed orremoved . The population size decreased to lesstha n 60adult s inth e subsequent summer but is recovering verywel l sincethe n (Wynhoff, unpublished data).

Procedure ofelectrophoresis Cellulose acetate electrophoresis was usedt o survey genetic polymorphism (system developed by Helena Laboratories (Hebert & Beaton 1993)).Al l insectswer e homogenised,eac h ina volum e of 0.04 ml buffer

solution of0.0 1 MTris ,0.00 1 MNaEDTA , 0.001 MMgCI 2,0.0 1 MMalei c acid and4m g NADP/100 mla t pH7.4 . Preparation and electrophoresis were performed ina coo l chamber. Details of electrophoresis aregive n intabl e 7.1a and 7.1b. For staining anaga r overlay was used.Stainin g recipes are given in Richardson (1986). Twenty-two different enzymes were tested,bu t mostwer e not scorable or were monomorph. Six loci (a-glycerophosphatase dehydrogenase (GPDH), Aconitase (Aeon), isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH),glucos e phospho-isomerase (PGI) and phosphoglucomutase (PGM)) showed ascorabl e banding pattern andwer e usedfo r genetic analysis (see table 7.3). During staining,th egel swer e photographed.Countin g of alleles took place onth e gels aswel l aso nth e photographs.

Table 7.1a: Enzyme loci analysed inthi s study with Enzyme Commission numbers, buffer system and voltage, time and starting place of electrophoresis.

Locus E.C. nr. voltage time starting running (V) (min) place buffer

PGM 2.7.5.1 200 45 cathodal TG PGI 5.3.1.9 200 40 central CP MDH 1.1.1.37 200 60 central P ACON 4.2.1.3 200 30 central TG IDH 1.1.1.42 200 60 central TM GPDH 1.1.1.8 150 75 cathodal P

139 Chapter 7:Genetic variation

Table 7.1b : Composition of running buffers.

running molarity PH composition of running buffer buffer (M)

TG 0.025 8.5 3.03 g/lTrismabase , 14.41 g/l glycine

CP 0.01 6.4 1.78 g/l Na2HP04.2H20, 0.48 g/l citric acid

P 0.02 7.0 2.06 g/l Na2HP04.2H20, 1.16 g/l NaH2P04.2H20 TM 0.05 7.8 6.06 g/l Trismabase, 2.32 g/l malic acid

Calculation ofgeneticalvariables Allelefrequencie s were calculated by means of Biosys-1(Swoffor t & Selander 1989).Standar d errorswer e calculated withth e SPSS package (Norusis 1993).Th e observed heterozygosity isderive dfro m Biosys-1, the expected heterozygosity was calculated byGD A (Lewis &Zayki n 1999).W e tested the difference between them with aX 2-testfo r goodness offi t (Biosys- 1).Th e standard error for bothvalue s was calculated according toWei r (1996). Forth e calculation ofth e Hardy-Weinberg equilibrium we used Genepop (Raymond & Rousset 1995). Linkage disequilibrium was also tested byth e Markov-chain method of Genepop. Significance was tested using sequential Bonferroni tests (Sokal& Rohl f 1995). For a pairwise testo f genetic differentiation we usedth e Markov-chain methodwit h 1000 dememorisations and 50 batches in Genepop (Raymond & Rousset 1995). For p-valuesth e Fishers'exac t testwa sused . F-statisticswer e calculated usingth e program GDA (Lewis &Zayki n 1999). Forth e meanvalu e per locusove r both populations we usedth e method described byWei r &Cockerha m (1984). The inbreeding coefficient

F|S was calculated indetai lfo r the locio f both populations within the species. Forth eestimatio n ofth e inbreeding coefficient, only thosewhic hwer e polymorphic in both populations within the species were included (for Maculinea teleius:IDH-2 , PGM,PG Ian dACO-K ;fo rM. nausithous: ACO-K,

PGM,ACO-A , PGIan d IDH-1).Th e difference between the F,svalue swa s tested withth eWilcoxo n signed rankstes tfo r paired samples (Sokal & Rohlf 1995).Al l F-statisticsvalue s are accompanied bythei r respective95 % confidence intervals estimated by bootstrapping with 1000replications .

Sample collectionfor electrophoresis The Moerputten populations inTh e Netherlands were sampled inbot h 1995an d 1996 because ofthei r limitedsize . Itwas , however, not possible to

140 Chapter 7:Genetic variation

collect many individuals of Maculinea teleiusi n 1996,s oth e sample size is smaller. The Polish Kostrza populations were sampled in 1996(tabl e 7.2). Butterflies in Polandwer e captured and put intoliqui d nitrogenfo r transport. Theywer e then stored ina freeze r at -80°C until analysis.Th e butterflies collected inTh e Netherlands were transported alive and immediately stored inth e freezer.

Table 7.2: Numbers of sampled butterflies of Maculinea nausithous and Maculinea teleius inth e respective years of sampling.

Site year Maculinea Maculinea nausithous teleius

Kostrza (P) 1996 41 50 Moerputten (NL) 1995 39 21 Moerputten (NL) 1996 19 0

Procedure andevaluation ofreintroduction Details ofth e reintroduction are given inWynhof f (1998a). Forth e estimation ofth e population sizetw o different methodswer e used.Firstly , in several, but not all,year s butterflies were marked and recaptured.Thes e datawer e usedt o estimate thedail y population sizea swel l asth e yearly population size by means oftw o methods:th e Minimal Number Alive-method andth e method ofJoll y (Begon 1979,Amle r era/. 1999). Inthi s way, alo w anda hig hestimat e of population size can begiven .Transec t countswithi n the Dutch Butterfly Network were performed inal lyear s (Van Swaay &Velin g 1996). Maculineateleius ha s been counted yearlyfro m 1990on .B y combiningtransec t counts and mark-recapture results,a nestimatio n ofth e yearly population size ispossible .Thus ,dat afro m theyear s when both methodswer e used,forme d the basisfo r alinea r regression,whic hwa sthe n usedt o extrapolate theyearl y population sizefo r yearswit h only transect counts. Since Maculineanausithous established itselfo n asit eothe r thanth e release site,w e selected additional newtransect s in 1995.

Estimation ofeffective population/census size Theeffectiv e population size isgenerall y defined asth e size ofa n ideal population that results ina give nvarianc e inallel efrequencie s or amount of inbreeding. Usually it issmalle r thanth e estimated population size. Inou r case it isno t possible to estimate the effective population size by means of

141 Chapter 7:Genetic variation the results ofth eelectrophoresis , because the sample size isto o small due toth e rareness ofth e butterflies. Furthermore the species appeared to beto o monomorpht o acquire reliable estimates (Schwartz efal. 1998) . Therefore we calculated whether a bottleneck effect wast o beexpecte d based onth e census data andth ese x ratio (Hartl& Clar k 1989,Avis e 1994, Maynard Smith 1998).W eca n usethes e population estimates to give upper boundst o the expected rateo f losso f genetic variation. Forth efirs t year, 1990, iti s assumed that alltranslocate d females hadalread y mated beforethe y were captured. Both species are protandrous.Afte r eclosion males almost immediately start searchingfo r females (Pfeiffer eral. 2000).The y even mate withfemale s who have notye t unfolded their wings. Furthermore, only the males mate several times,whil e thefemale s usually matejus t once. Duet o the high population density atth e source site, it isver y likely that all females matedwit hdifferen t males. The meaneffectiv e population size (based oncensu s data) over a period ofyear s isestimate d bycalculatin g the harmonic mean over all effective census sizeso f allyear s between 1990an d 1995(Avis e 1994).W e assume random mating and anequa l chance for all individuals for progeny inth e next generation.Thi sassumptio n isviolate d inth efirs t and lastday so fth e flight periodwhe nth e sex-ratio is biasedt oon e ofth e genders, but it is likely to be truefo r mosto fth etime .A low bound ofth e effective census Nemjnsiz e is basedo nth e Minimal NumberAliv e data,a maximal effective census size

Nemaxo nth eJoll y estimates. Maculinea teleius was marked and recaptured in 1990, 1991, 1992an d 1995. For the intermittent yearswhe n only transect counts took place,th e population sizewa s estimated by interpolation based on linear regression (Sokal& Rohl f 1995). For the sex ratio,th e mean value ofth eyear s before and after was calculated. For Maculinea nausithous, the same procedurewa sfollowed . However, asth etransec t counts started later and onothe r places thenwher e mark-recapture took place,th e error inou r estimate is larger. Mark-release-recapture studies of Maculinea nausithous took place in 1990, 1992, 1993an d 1995.

Fromth eeffectiv e census size,th e upper bounds ofth e chance of loosing heterozygosity through genetic drift can becalculate d by means ofth e followingformula :

Hret =(1-(1/2Ne)) '

with : Hret =retaine d heterozygosity after bottleneck Ne =effectiv e census size t =numbe r of generations

142 •M ••• Chapter 7:Genetic variation

Results

Genetic variation of Maculineateleiu s andM . nausithouspopulations Thegeneti cvariatio n isver y similar between speciesan d populations. The mostcommo n alleles areth e same ineac h population.Allel e frequencies are also similar. There isonl y limited evidencefo r losso f alleles. ForM. teleius onlyth e locus IDH-2 inth e Kostrza-populations segregated for three alleles, PGM, PGI,ACO- Aan dACO- Kfo r twoalleles ,whil e IDH-1, MDH-1, MDH-3 and GPDHwer e monomorphic (appendix 7.1). Inth e Moerputten-population the rareallele s of IDH-2 andACO- Aappea r to have been lost.Whil e 11 different genotypes were detected inth e Kostrza-population, only6 genotypes werefoun d inth e Moerputten-population. However, the sample sizewa s only about half as large. So,fro m Moerputten,som eo fth e apparent loss may bea n artefact.

Inth e case ofM. nausithousonl yth e loci MDH-1 and IDH-2ar e monomorphic over both populations. PGIseparate d withthre e alleles,al lth e other enzymes withtwo .On e allele of MDH-2 ando f GPDH are notfoun d in the Moerputten population. InMDH- 3a rar e allele wasfoun d inth e Moerputten sample but not inth e Kostrza sample (appendix 7.1). Twenty genotypeswer e detected ineac hpopulation . A reduction inth e number of alleles can beexpecte d asth e result ofth e reintroduction bottleneck. One locusfo rM. teleiusan dtw ofo rM. nausithous have becomefixe dfo r the common allelefiv eyear s after reintroduction.Th e meanheterozygosit y islo wi nal lfou r analysed populations (appendix 7.2). There were nosignifican t differences between source and reintroduced population exceptfo r onelocus .Th e heterozygosity-values forM. nausithous were slightly higher thanfo r M. teleius.Overal l withth e possible exceptiono f a limited loss of rare alleles ineac h species there is nolos so fgeneti c variation. Only one locus ineac hspecie swa s not in Hardy-Weinberg-proportion for a single sample.Th e IDH-2 locus isno t inequilibriu m inth e Kostrza population ofM. teleius, while the MDH-3 locus deviates inth e Moerputten sample ofM. nausithous.N olinkag e disequilibrium wasfoun d inan yo fth e population samples of bothspecies .

Populationdevelopment afterreintroduction Fromth e regression analysis oftransec t counts and mark-recapture studies ofM. teleiusa reliable approximation of thetota l population size per

year couldb eyielde d (Nmin=a*Ntrans,a=16.3 ,SE a=0.74, p<0,0001,1^=0.99;

Nmax=b* Ntrans,b=24.4 ,SE„=1.3 , p<0.0001, r^O.99) (figure 7.2). The minimal

143 Chapter 7:Genetic variation and maximal population sizes ofM. teleiusan d M.nausithous are presented infigur e 7.3.Th e population structureo fM. teleiuswa s quite similar ineac h yearfro m 1991t o1995 ,whe n the sampling for the genetic analysis took place.Th e population increased from the reintroduction event upunti l 1992. Then, possibly duet o overexploitation ofth e ant nests,th e butterfly population decreased toabou t 300 individuals. Over allyear s on average 62.7(± 7.7)% ofth ecapture d butterflies were females.Th e mean effective population size derivedfro m the census size isa s higha s 94.5 (±2.5) % of thetota l censussize .

The populationo fM. nausithoussuffere d asecon d potential bottlenecki n 1991whe nonl y 8 individuals were encountered.Sinc ethe nth e population increased approximately linearly. The sex ratiowa sclos e to equality (59.7(± 7.8)% females).Th e meaneffectiv e census size isagai nquit e high: 95.9 (± 4.5)%o fth e population size. However, duet oth e second bottleneck, the meaneffectiv e population size isonl y 38t o4 0 individuals.A detailed overview ofth e population development ofMaculinea teleiusan d M. nausithous isgive n inWynhof f (1998a).

N 1250 x Minimum population size (MNA)

• Maximur npopulatio nsiz e (Jolly) 3 m^r 9-1000 o Regressionlin e minimum population size

Regression line maximum population size

^^ x.--"'

^s^ • . -* * \S^ x.. - ""

^/"^.- -'" x -*

Figure 7.2: Regression line oftransec t counts and mark-recapture studies of Maculinea teleius.

144 Chapter 7:Genetic variation

Table 7.3:Predicte d effect of bottleneck passed byth e reintroduced populations of

Maculinea teleius and M. nausithous in 1990. Ne:effectiv e census size; Hret: heterozygosity retained after bottleneck.

Maculinea teleius Maculinea nausithous

Nr. of introduced females 53 48 Nr. of introduced males 33 22 Ne (2x females) 106 96 H,„, 0.995 0.995

Predictedeffect of reintroduction bottleneck Based onth e number oftranslocate d individuals,th e expected maximum proportiono f heterozygosity retained afterth eartificiall y introduced bottleneck can beestimate d following the above mentioned formula (table 7.3). Weals o estimated the expected lossfo rth e casetha t allfemale s would have matedwit hth esam e male,whic h isver y unlikely. Furthermore,a limited number offemale s may have been ablet o oviposit. However, we observed many ovipositions from different females immediately after release.

Year

Figure 7.3: Population development (intota l population numbers per year) of Maculinea teleius (circles) and Maculinea nausithous (triangles) after reintroduction in 1990. open symbol: minimum population size closed symbol: maximum population size

145 Chapter 7:Genetic variation

Thus,th e number of translocated individuals islikel y to have been high enough to prevent any significant losso f genetic variation duet oth e reintroduction bottleneck. However, wewishe d to check this usingth e direct estimates of genetic polymorphism.

Genetic differentiation ofKostrza andMoerputten-populations: the possible bottleneckeffect Although being separated for onlyfiv e generations the populations of Kostrza and Moerputten have already become differentiated atth eACO- K locus (Fisher's Exact p=0.0062 for M. nausithousan d p=0.0003fo rM.

teleius).A tthi s locusth e FST- value asa measure of population subdivision is much higher compared thevalu efoun d forth e other loci (appendix 7.3). Over all locith e populations do not show significant differentiation, Nei's genetic distance between the populations isfo r M. teleiusestimate d at0.00 3an dfo r M. nausithous at 0.004. Thus the data suggest that somedirec t selection has occurred on polymorphism atth eACO- K locuso rtha t there isstron g linkage disequilibrium andhitch-hiking .

The FIS value represents deviationfro m local panmixis anda s such canb e interpreted asa n inbreeding coefficient. Ifa populatio n has passeda

bottleneck the F,svalu e isexpecte d to increase. For both species the mean

F|Svalue s calculated forth e polymorphic loci present insourc e andfounde r population,ar e not significantly different from zero inth e Polish source populations. Inth e Moerputten populations,the y were negative and differed significantly from zero (figure 7.4). However, no significant difference toth e

source population's F,svalue s wasfoun d (Wilcoxon paired samples: p=0.068 forM. teleiusan d p=0.225 forM. nausithous). Theobserve d heterozygosity was higher inth e Moerputten population compared toth e Kostrza population,

though not significantly. Ifth e F,s valuesfo r both species are pooled (to estimate anoveral l inbreeding effect ofth e reintroduction), there isa n overall decrease ininbreedin g coefficient (Wilcoxon paired samples p=0.038). Exceptfo rM. teleiusa tth e Moerputten site (r=-0.999, p=0.001,no t

correlated.A decrease in F,si ncombinatio n with an increase in heterozygosity can beachieve d byoutbreedin g or heterozygous advantage.

Discussion

Effectsof reintroduction bottleneck Whena smal l group of animals or plants isisolate d forwhateve r reason from their ancestral population,a divergenc e ofth e separated gene pools is

146 Chapter 7:Genetic variation

Figure 7.4:Change s in inbreeding coefficient F,so f source population and new population of Maculinea teleius (left side) and M. nausithous (right side) after five years of separation since reintroduction in 1990. Bars represent 95% confidence intervals estimated by bootstrapping (1000 replications). K = Kostrza (source population), MP = Moerputten (reintroduced population)

expected,wit hth e smaller or more isolated group tendingt o loosevariation . Saccheri efal. (1998 )wer e able tosho wtha t this process isals o detectable inth efiel dfo rth e butterfly species Melitaeacinxia. After asever e reduction ineffectiv e population size,geneti c drift is an important evolutionary force and rare alleles tendt o be lostthroug h chance. However, a bottleneck effect isonl y detectable whenth e bottleneck has reduced the size ofth e breeding population usually severely andespeciall y whenthi s has persistedove r n= 4), F,san d heterozygosity were several generations (Saccheri efal. 1996 , Van Oosterhout efal. 2000,Va n Hooft 2001). For Maculinea nausithous and M.teleius th e separation ofgen e pools since reintroduction has enduredfo r onlyfiv e generations atth e moment of sampling. Large genome-wide genetic changes are unlikely after such a limitedtim e span inregion s where ecological conditions are supposed to besimila r andwhe nth e effective number oftranslocate d individuals wasquit e high.Th e analysed allozymes indicate that the losses have indeed been mild andwit h respect tothis ,th e reintroduction did not represent asever e bottleneck toth e populations. Loss of heterozygosity, aswa sfoun dfo r the butterfly Proclossiana eunomia (Barascud efal. 1999) , has probably been prevented byth e high number of

147 •• •• Chapter 7:Genetic variation

founders andth esuccessful! ,rathe r rapid establishment. If nearly all ofth e founding females laidviabl e eggs any expected loss of genetic variation would be minimal and be impossible to detect using limited electrophoretic data. However, ifonl y afe w individuals were successful inleavin g offspring and/or subsequent generations were of very smalleffectiv e size,th e genetic effects would be likely to bedetectable . Furthermore, inon e experimental study, post-bottlenecked populations showed an increase in heterozygosity through heterozygous advantage, eventhoug h alleles had been lost by suffering astron g bottleneck (Leberg 1992). Both reintroduced populations of Maculinea, however, have lost several rare alleles. Even inM. nausithous, which isthough t to have experienced smaller population sizes during establishment, ofth esi x alleles detected ata frequenc y of <10% inPoland , four were detected inth e Moerputten population after 1991(an da n additional rareallel e whichwa s undetected inth e Polish sample).Thes e data also indicate that inthi s species the effective population in 1991, the year after release,wa s higher than the 8 butterflies observed inth e field.

Implications oflow genetic variation Both studied Maculinea species show a low but notexceptionall y low level of genetic variation as compared totha tfoun d for polymorphic enzyme locio f other butterfly species (Barascud etal. 1999; Napolitano & Descimon 1994; Goulson 1993;Brooke s etal. 1997;Gadeber g & Boomsma 1998;Va n Dongen etal. 1998;Clark e &O'Dwyer2000 ; Figurny-Puchalska etal. 2000). In reality, genetic variation might even be lower because there isa bias inth e allozymes chosen for the study: most monomorphic enzyme systems have beenomitted . Having sufficient genetic variation isofte n thought to bea prerequisite to successfully translocating a parto f apopulatio n toanothe r site and retaining potentialfo r adaptive evolution to changing conditions (Hunter 1996).Thus , eventhoug h they are seriously threatened (Van Swaay &Warre n 1999; Wynhoff 1998b),Maculinea species might intrinsically not bever y suitablefo r reintroduction. Figurny-Puchalska efa/ . (2000) alsofoun d low levels of genetic variation inth e Kostrza-populations, butothe r Polish and Russian populations were more polymorphic. It is possible that thesourc e population could have been subject to arelativel y recent bottleneck event, as has been indicated for other butterfly species too (Meglecz efal. 1999) . However, the success ofth e reintroduction at Moerputten shows that eventhoug h both species show alo w level of polymorphism usingth e limited methodology of allozym electrophoresis, itwa s not aconstrain t to their successful establishment. Inth eWisl avalle y both Maculineaspecie s are abundant but M.teleius occurs in higher numbers than M.nausithous. Henc e basedo n

148 Chapter 7:Genetic variation

census sizesfo r M. teleiusmor evariatio nwoul d beexpecte d but lesswa s found. Perhaps,du et o itsspecialise d ecology,thi s species ismor e sensitive to catastrophes. M.nausithous seems to be more robust: eventhoug h itha s experienced smaller numbers, genetic losses have been less compared to M. teleius.I n Poland,thi s robustness was expressed inth e lack of genetic differentiation with increasing spatial scale (Figurny-Puchalska efal. 2000).

Onth eothe r hand alo w level of heterozygosity cannot always be considered a problem for theviabilit y of populations orfo rth efitnes s of individuals, asther e are many rare andcommo n species lacking ahig h level of heterozygosity while other rare or endangered species have been shown to have normal or high levels of heterozygosity (Avise 1994). So ingeneral ,a prediction ofsurviva l of bottlenecked populations merely based onthei r genetical variation isno t possible. Dunham etal. (1999 ) suggest that iti s likely that species with limitedwithi n population variability, or non-equilibrium population structure may bewel l adapted to persist inth efac e of extreme isolation or small population size.Thi s may beth e casefo r species that inhabit naturally isolated,bu t relatively stable habitats. Obligate ant parasite butterfly species,whic h are adapted to specific habitat conditions and cannot survive ina changin g environment, sucha s M.teleius livin g in Myrmica scabrinodis nests onwe t meadows may have such adaptations aswell . The example ofth e reintroduction ofth e beaver to Sweden shows that a species with low genetic variation can survive andeve n spread sucessfully over many years andgeneration s (Ellegren etal. 1993).Thi s case ofth e /Wacu/inea-butterfliesals o demonstrates through the increase in population size,th e balanced sex ratio andth e low chance of loosing heterozygosity in thefounde r population,tha t the Kostrza populations as source population for a reintroduction projectwa s agoo dchoice ,a t least inth eshor tterm . Iti s probably most important toenabl e agoo dstar tfo r areintroductio n project by releasing manyfounde r individuals and byfindin g a release sitewhic h fullfills allecologica l demands of aspecies .

Possibleselection on the founder populations Although thevariatio n inthes e Maculinea species is rather low,a t leastfo r a very limitedgrou p of enzyme loci,ther e is no reasont o suppose that this will impede adaptive evolution in response toth e different ecological conditions inTh e Netherlands. Indeedw eobserve d evidencefo r sucha process bothfo r oneo fth eenzym e loci (ACO-K)an dfo r phenology. Changes inth e allozymefrequencie s were notdramati c and only occurred at one locus.A t theACO- K locusth e populations of Maculinea nausithous and M.teleius sho wa significan t differentiation between the ancestral Kostrza-

149 Chapter 7:Genetic variation

population andth e newlyfounde d Moerputten population.Th e population studies atth e reintroduction site also revealed that there was a gradual forward shift inth efligh t periodfro m ofM. teleiusan dt oa lesser extent inM. nausithous, withth efligh t period inth e Netherlands peaking earlier than in Poland andtha nth e historical Dutch populations (Wynhoff 1998a).Th e observation ofa gradua l shift suggests that this isunlikel y to have resulted purely from a response inphenotypi c plasticity. Furthermore,th e mean minimal lifetim e percapture d individual hasdecrease d strongly inth e Moerputten population of both species (Wynhoff 1998a).Thes e changes point inth edirectio n of different ecological and environmental conditions and the potential for selection.Condition s atth e new sitewer e probably quite different from those inth eWisla-valle y in Polandfavourin g adaptation toth e newenvironment . Selection has also been shownt o influence enzyme polymorphism inothe r butterfly species (McKechnie efa/ . 1975,Wat t 1977, Watt era/. 1986,Goulso n 1993).

Genetic differentiation betweenpopulations Within Europe,Maculinea nausithousi s more common than M. teleiusan d the population size isals o often much higher. Furthermore, M. nausithous can survive for long periods with small populations of lesstha n 100 individuals on marginal habitats such as roadverge s and canal borders (Settele 1998;Wynhof f 1998b)whil e M. teleiusi s restricted to larger and less dynamic sites. Populations ofM. nausithous are considered to be more connected with higher rates of interchange. Hence, inth e source population

the FST value, representing the variance inallel efrequencie s between subpopulations andtherefor e beinga measure of populationsubdivision , should be lower inM. nausithous as compared to M.teleius. Thi s is indeed confirmed byou r analysis.

After five years of separation, nosignifican t differentiation expressed in FST value between source andfounde r population ineithe r species could be detected. Ifth e differentiating Aco-K isexclude d from the F-statistics, values are closer tozero , butth e conclusion still holds.O na higher regional level

significant population structure in both species bya relativel y high FST value has been suggested (Figumy-Puchalska etal. 2000) , butther ewa s no isolation bydistance .

Effectsof inbreeding Small populations are especially pronet o losso f genetic variation by

inbreeding,whic h can beshow n bya significan t positive FIS value.Th e probability of matings between relatives increases insmalle r randomly

mating populations. For both Maculinea species,th e significant negativeF, s

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inth e comparably smaller, newlyfounde d Moerputten population indicates that inbreeding may beavoided .Thi s is unexpected and atth e sametim e could perhaps be important asth efounde r populations are completely

isolated.Apar tfro m chance, negative F,svalue s can beexplaine d intw o ways: (i)outbreeding : relatives avoid mating witheac h other, (ii) heterozygote advantage: inth e newenvironmen t the heterozygote hasa higher fitness compared toth e homozygote. Ifoutbreedin g or heterozygous

advantage were anexplanatio n for thedecreas e in F,svalue ,i t should be accompanied bya n increase in heterozygosity. In bothspecie s thiswa s not

found. Furthermore, inbreeding affects all loci similarly, hence FIS values should besimila r atal l loci ina give n population.W edi d not observe sucha

pattern inan y ofth e populations.Th e decrease in FIS values isconsisten t in both species,wit hth e steepest decrease inth e lociwit hth e highestvalue . Whenw e acceptselectio n as responsible forth echange s inth e Moerputten population compared toth e Kostrza population,the nth eAco- K allele hast o beexclude d fromth e F-statistics. Thetren dthe n remains similarfo rM. nauithous. Inth ecas e ofM. teleiusi nth e Moerputten population,th e

detected differences areonl y very weak. Thus the F,svalue s may hinta t some active or inactive avoidance of inbreeding inth e Moerputten population,althoug h the mechanism isunknown .

Conclusions

We concludetha tth e reintroduced populations of Maculinea nausithous and M.teleius i nth e Moerputten did not passthroug h a bottleneck interm s ofa low number offounde r individuals.Th e low levels of genetic variation of the source populations didno t have a negative impacto nth e settling and subsequent increase ofth efounde r populations.Th e number of translocated founder individuals andth e quality ofth e release site have ahighe r impacto n success thanth e genetic constitution ofth efounders . The differentiation between source andfounde r population since reintroduction, inth eallozyme s aswel l as infligh t period and life time,ar e probably duet oselection .I n reintroduction projects withthi s type of species, it is important to create as soona s possible a metapopulation atth e release site. Inthi swa y stochastic extinction ofsubpopulation s can becompensate d for because inth e metapopulation,gen e exchange between subpopulations is possible.

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Acknowledgement

It isa pleasure to express ourgratitud e to Mariska Hartean d Simon Plat who helped IWcollectin g butterflies in Poland.W ear egratefu l to Edyta Figurny, Ewaan d Michal Woyciechowski andth e Sieniawski familyfo r their hospitality and help in Poland.Th e Polish Ministry of Environment andth e Dutch State Forestry Service kindly allowed ust o collect butterflies for this study.A special thank you goest o Dr. Maurice Franssen (Department of Organic Chemistry, Wageningen University) who kindly allowed ust o use his -80°C-freezer.Thi swa s crucial for the success ofth e study. Pirnva n Hooft, Peter deJon g and CorZonnevel d gave critical and stimulating remarks to improve the manuscript. The expedition toth e Polish siteswa sfunde d byth e Uyttenboogaart- EliasenStichting .

152 Chapter 7:Genetic variation

Appendix 7.1: Allele frequencies of Maculinea teleius and M. nausithous at Kostrza and Moerputten. (N)= sample size, nA= tota l number of alleles, nL=mea n number of alleles per locus, H = mean heterozygosity per locus (direct count), PDC= mean percentage of polymorphic loci (no criterion), P95= mean percentage of polymorphic loci (95%criterion) .

Maculinea teleius Maculinea nausithous

Locus Allele Moerputten Kostrza Moerputten Kostrza

frequency±SE frequency±SE frequency±SE frequency±SE PGM (N) 21 50 58 41 A 0 0 0.276±0.042 0.220±0.046 B 0.952±0.033 0.990±0.010 0.724±0.042 0.780±0.046 C 0.048±0.033 0.010±0.010 0 0

PGI (N) 21 48 58 41 A 0.071±0.04 0 0.052±0.022 0.043+0.019 0.049±0.024 B 0.929±0.040 0.948±0.026 0.914±0.026 0.927±0.029 C 0 0 0.043±0.019 0.024±0.017

ACO-K (N) 21 50 58 41 A 0.190±0.061 0.040±0.020 0.957±0.019 0.793+0.045 B 0.810±0.061 0.960±0.020 0.043±0.019 0.207±0.045

ACO-A (N) 21 50 58 41 A 0 0.090±0.029 0.086±0.026 0.049±0.024 B 1.000 0.910±0.029 0.914±0.026 0.951±0.02 4

IDH-2 (N) 21 50 58 41 A 0.024±0.024 0.030±0.017 0 0 B 0.976±0.024 0.960±0.017 1.000 1.000 C 0 0.010±0.010 0 0

IDH-1 (N) 21 50 58 40 A 1.000 1.000 0.922±0.025 0.927±0.033 B 0 0 0.078±0.025 0.073±0.029

MDH-3 (N) 21 50 58 41 A 1.000 1.000 0.017±0.012 0 B 0 0 0.983±0.012 1.000

MDH-2 (N) 21 50 58 41 A 0 0 0 0 B 1.000 1.000 1.000 0.988±0.012 C 0 0 0 0.012±0.012

GPDH (N) 21 50 57 40 A 1.000 1.000 1.000 0.988±0.017 B 0 0 0 0.013±0.012

MDH-1 (N) 21 50 58 41 A 1.000 1.000 1.000 1.000 14 16 17 18 1.40±0.16 1.60±0.22 1.70±0.21 1.80±0.20 H 0.067±0.038 0.044±0.019 0.100±0.042 0.100±0.035

•DC 40 50 60 70 P95 20 20 40 40

153 Chapter 7:Genetic variation

Appendix 7.2: Observed (Hobs)an d expected heterozygosity (He>(p)pe r locusfo r Maculinea teleius andM. nausithous. (Total number of alleles: 10).* = no testavailable .

Hobs Hexp 2 Site locus mean±SD mean±SD x df P

Maculinea teleius Moerputten PGI 0.143±0.076 0.136±0.066 0.081 1 0.776 PGM 0.095±0.064 0.093±0.05 9 0.026 1 0.873 IDH-2 0.048±0.047 0.048±0.04 5 0.000 1 1.000 ACO-K 0.38U0.106 0.316±0.06 7 0.998 1 0.318 mean 0.067±0.038 0.059±0.030 Kostrza PGI 0.104±0.044 0.100±0.040 0.115 1 0.735 PGM 0.020±0.020 0.020+0.020 0.000 1 1.000 IDH-2 0.06010.034 0.078±0.041 * ACO-A 0.180+0.054 0.165±0.045 0.431 1 0.521 ACO-K 0.080±0.038 0.078±0.035 0.064 1 0.800 mean 0.088±0.018 0.044±0.017

Maculinea nausithous Moerputten PGI 0.172±0.050 0.163±0.044 0.461 1 0.927 PGM 0.414±0.065 0.403±0.037 0.043 1 0.836 MDH-3 O.OOOiO.OOO 0. 034±0.033 * IDH-1 0.155±0.048 0.144±0.040 0.362 1 0.547 ACO-A 0.172±0.050 0.159±0.041 0.461 1 0.497 ACO-K 0.086±0.037 0.083±0.034 0.093 1 0.760 mean 0.100±0.023 0.099±0.016 Kostrza PGI 0.146±0.048 0.140±0.050 0.211 3 0.976 PGM 0.293±0.071 0.347±0.056 1.050 0.305 MDH-2 0.024±0.024 0.024±0.023 0.000 1.000 IDH-1 0.146±0.056 0.141±0.048 0.211 0.646 ACO-A 0.098±0.046 0.094±0.042 0.080 0.777 ACO-K 0.268±0.069 0.333±0.057 1.619 0.203 GPDH 0.025±0.025 0.025±0.024 0.000 1.000 mean 0.100±0.019 0.110+0.017

154 Chapter 7: Genetic variation

Appendix 7.3: Estimates ofWright' s F-statistics. The mean values per locus are given.Th e

confidence intervals of the mean value over all loci are obtained by bootstrapping. The FISvalue s are presented infigur e 7.4.

Locus FIT FST

Maculinea teleius PGM -0.0043 0.0174 IDH-2 0.1742 -0.0175 PGI -0.0621 -0.0131 ACO-A -0.0293 0.0511 ACO-K -0.0085 0.1225

mean -0.0002 0.0489 Bootstrapping over loci (C.I.95% ) upper 0.0822 0.0990 lower -0.0414 -0.0114

Maculinea nausithous MDH-2 0.0022 0.0044 PGI -0.0642 -0.0081 MDH-3 1.0000 -0.0062 PGM 0.0406 -0.0025 ACO-A -0.0705 0.0010 ACO-K 0.2353 0.1172 GPDH 0.0023 0.0046 IDH-1 -0.0922 -0.0096

mean 0.0579 0.0202 Bootstrapping over loci (C.I.95% ) upper 0.1845 0.0751 lower -0.0494 -0.0067

155 Chapter 8: Synthesis

Synthesis

Partsar e publishedas :

Wynhoff I,CA Mva n Swaay,AM H Brunsting& JGva n der Made (2001d): Conservation of Maculinea butterflies at landscape level. Proceedings ofth e Section Experimental &Applie d Entomology, NEVAmsterda m 12: 135-141.

157 Chapter 8: Synthesis

158 Chapter 8: Synthesis

Onth ewar m evening of July 30th, 1990,th efirs t official butterfly reintroduction inTh e Netherlands took place.Ja nva nde r Made,Chri sva n Swaay, Ivo Raemakersan d Irelease d Maculinea teleius and Maculinea nausithous butterflies onth e meadows ofth e nature reserve Moerputten. Now,te ngeneration s later, afterwar man dwe tsummer s have passed,th e time has comet o make anevaluation .Wa sth e reintroduction ofthes e butterflies asucces s or afailure , is reintroduction a usefulan d applicable technique in nature conservation? Are Maculineabutterflie s still under threat of extinction? What havew e learned about their ecologywhil e monitoring and studying the newly established populations? Inthi s chapter Iwil l putal l the small pieces ofth e Maculinea storytogethe r so ast o obtain amor e complete picture.

The successof the reintroduction

After the reintroduction,th e newlyfounde d populations of Maculinea teleius and M.nausithous behave d differently. The introduced population of M.teleius expande d during thefirs tthre e years.Then ,adul t numbers decreased and stabilised onth e occupied meadow BW(figur e 6.1, figure 7.3). This process happened twice,sinc e in 1995th e meadow was accidentally mown inth e flight period. Itresulte d inth e reduction ofadult s in 1996t oth e level ofth efounde r population atth e reintroduction. Noothe r meadow wasoccupied ,sinc ether e was almost no long distance dispersal after 1993. Occasional dispersal was observed butwa s notfollowe d upb y the establishment of ane w subpopulation.Apparently , inth e existing conditions at meadow BW,th e yearly population size hasfoun d its limitwit h about 300 to40 0 individuals (Wynhoff 1998b , chapter 3).A n increase in population size is most likely only possible through dispersal and establishment at unoccupied patches. Incontrast , the newly established population of M. nausithous declined in numbers inth eyea r after the reintroduction butexpande d later. Aftera considerable increase in numbers,thi s species colonised the roadverge s at the southern side ofth e nature reserve. Later, athir d subpopulation was established ata distanc e of 5kilometer s toth ewes t (Wynhoff 1998b , chapter 3). InTh e Netherlands, a reintroduction of butterfly species is considered successful whenth e introduced population can persistfo r at least fiveyear s without further intervention (Wynhoff &Va n Swaay 1995). Ina ne w edition ofth e Red List, both Maculineateleius and M.nausithous woul d not beliste d as extinct species any more.

159 Chapter 8: Synthesis

Unfortunately, adult numbers of Maculineanausithous areno w decreasing,a declin e which began afe w years ago.A tth e embankment, this isdu et ovegetatio n succession through which the occupied patch deteriorates while there is nosuitabl e location within reach ofth e butterflies available to relocate the population.O nth e road verges,th e decline isclearl y duet o human activities. Management by (local) authorities and local residents and reconstructions ofth everge s have destroyed large parts ofth e vegetationwhic h reduced survival ofth e reintroduced species andth e host ants.Thi s development clearly shows,tha t the reintroduction of rare butterfly species into agricultural landscapes isrisky . Localauthoritie s and residents should beinforme d andthei r full support should begained .Afte r all, they shapeth e habitat inwhic hth e newcomers havet osurviv e (Oostermeijer& Wynhoff 1996,Wynhof f &Janse n 2000).

Inth e Moerputten nature reserve, Maculineateleius an d M. nausithous occur spatially separated,whil e in many regions they sympatrically inhabit the same meadow,fo r example inth e Kostrza source population (Ebert& Rennwald 1991, Wynhoff 1998b, Figurny &Woyciechowks i 1998).A tth e reintroduction site,a typica l habitat of Maculinea teleius consists of Molinietalia vegetation with astron g Junco-Molinion aspect. Itconsist s ofa rather short cover upt oa height of about 50c mo na comparabl y poor soil with a high density ofth e host ant Myrmica scabrinodis. The nutrient poor vegetation communities belong to Junco-Molinion with high abundances of Succisapratensis an dJuncus conglomeratus. Locally atth e meadow BW, where there are high abundances of Cirsium dissectum and Carexpanicea, thevegetatio n can beclassifie d as Cirsio dissecti-Molinietum. InTh e Netherlands, it isfoun d on moist, slightly acidic soil,tha t is inundated during wintertime . Within these vegetation types,th e host plant Sanguisorba officinalis can occur in high densities witho n average 10(SD=7 ) plants with amea n of 17 (SD=15)flowerhead s per square meter onth e meadow BW. Onth e other meadows atth e southern side of Moerputten,th edensit y of plants aswel la s flowerheads is higher. However, density and quality of hostants ' nests are the mainfactor s determining butterfly behaviour, butterfly density and survival ofth e larval instars (see chapter 4t o 6).Ther e can bea considerable variation inhos t ant nestdensit ywithi n and between sites.Th e source population in Kostrza persisted on Myrmica scabrinodisnes t densities between 0.1 and 1.2 nests per square meter. At the Dutch site,densitie s are approximately thesam ewit h 0.80 nests per square meter (Wynhoff 1992).A t the continental scale,th e most important host antspecie s is Myrmica

160 Chapter 8: Synthesis scabrinodis, butals o Myrmica rubraca n beexploite d as asecondar y host (Chardon &d e Boer 1994,Eber t & Rennwald 1991, Thomas etal. 1989, Figurny, pers.comm.) .A t the reintroduction site, however, Maculinea teleius appaers to be restricted to Myrmicascabrinodis. Asth e behaviour of Maculineateleius caterpillar s inth e ant nests issimila r to Maculinea anon (Thomas & Elmes 1998),a highdensit y of relatively small nests ofMyrmica scabrinodisprobabl y yields the highestsurvival . Thevegetatio n onth e meadows WH, AH,PN ,PZ , DHan d HvB consists of amozai co f Molinietalia andArrhenateretalia types with patches rich in herbs or rich insedge s and grasses. Low lying locations under influence of base-rich seepage show aspects of Calthionpalustris wit h Lychnisflos-cuculi, Rhinanthus angustifolius and Lotusuliginosus. On higher anddrie r sitesth etypica l hay-communities Arrhenaterionelatioris and Alopecurionpratensis occu r closely together. The density of plants and flowerheads of Sanguisorba officinalisi s higher thano nth e occupied meadow BW,however , the host ant Myrmicascabrinodis occurs only locally andwit h alowe r ant nestdensit y onthes e meadows. Ifth eoccurenc e of Maculineateleius an d M.nausithous wer e only dependent onth e availability ofth e preferred flowerbuds ina n acceptable phenological stage duringth e flight period,a s proposed byThoma s & Elmes (2001), both butterfly species should bethrivin g there sympatrically.

Inth e same nature reserve, Maculinea nausithousprefer s different conditions than Maculineateleius does.O n preferred sites,a species-poor Arrhenaterionelatioris vegetation ,als owit h characteristics ofArtemisietea, is found. Thevegetatio n structure istalle r and less open compared to siteswit h Maculineateleius. O nth e roadverges MPPan d RD,thes e latter types also occur, eventhoug h typical herb species are missing and locally vegetations resemble Cynosurion and Plantaginetea majoris. The communities from the railway embankment SD includeArrhenateretalia patche s with typical members ofth eArtemisietea an d Galio-Urticetea, such as Urticadioica an d Cirsium arvense.Sanguisorba officinalis occurs inal lthes e vegetationtypes , albeit invariou s abundances (Ten Oever & Brongers 1994,Zuidhof f etal. 1996, Schaffers &Sykor a 2000). Onth eembankment , a meandensit y of 3.7 (SD=3.9) plants wasfound ,wit h as much as42. 5 (SD=41.8)flowerhead s per square meter. Onth e road verges,fewe r plants but moreflowerhead s were available for the butterflies (plants: 8.6±6.5, flowerheads 77.8±53.7). Whilefo r Maculinea rebelia positiv e relationship between the population size ofth e host plant Gentiana cruciataan d that of the butterfly wasfoun d (Kery etal. 2001, Elmes etal. 1996,Hochber g etal. 1994),suc ha relationship

161 Chapter 8: Synthesis

does not existfo r Maculinea teleiusan d M.nausithous an d their respective hostplant (chapter 5). At the site naturally colonised by Maculineanausithous, the highest nestdensit y of its host ant Myrmica rubrawa sfound ,wit h upt o6 nests per square meter. Onal l locations colonised bythi s butterfly species,Myrmica scabrinodis wasals o found. However, siteswit h only the latter ant species were not colonised.A tth e Kostrza source population site,th e mean density of Myrmica rubrai s0. 4 nests per square meter. However, attha t particular location, both antspecie s co-occur. Thesuccessfu l reintroduction also shows that the communication between the "Polish"caterpillar s andth e "Dutch"ant s isfunctioning . Lycaenid larvaewhic h enter ant nestst o predate onan t brood could never dos o without havingth eappropriat e communication means (Fiedler etal. 1996) . The caterpillars of Maculinea rebel!, anadvance d predator ofMyrmica schencki,produc e surface chemicals which enable them to be mistaken for their host ants' brood and betake n toth e nest.Th e mimetic chemicals resemble those employed by Myrmica to recognize conspecifics and arever y similar tothos e ofth e specific host ant species (Akino etal. 1999) .Fo r another advanced parasite,Maculinea alcon, difference s inthes e pheromone-likechemical s were found between populations parasitising on different Myrmicahos t ant species in Denmark (Nash,pers . comm.).Thi s phenomenon suggests adaption at local level.Obviously , inth e primitive parasites Maculinea teleiusan d Maculinea nausithous, pheromoneso f parasite and hostan t are similar within large parts ofthei r range.Th e same can be concluded for Danish Maculinea arionbutterflie s which were released intoa n English sitewit h Myrmicasabuleti (Thoma s 1989, 1991, 1995). Furthermore,th e lack of parasitoids inth e reintroduced butterfly populations might also give anadditona l reasonfo rth e rapid increase of adult numbers. However, inth e large source populations,th e impact of Maculinea specific parasitoids issmal l or restricted to certain years (Chardon & De Boer 1994), andw e don't think that the parasitoidfre e space has contributed much towards the success ofth e reintroduction.

Oviposition behaviourand habitat selection

An eggo f a Maculineabutterfl y is not beingdeposite d untilth e female has selected asuitabl e oviposition site.Th e intention to deposit aneg g is already detectable inth ewa y afemal e flies:sh eflutter s slowly and low inth e vegetation between theflowerhead s that emergefro mth edens e vegetation

162 Chapter 8: Synthesis cover. Thefligh t direction changes frequently. Often,a flowerhea d of Sanguisorba officinalisi na nacceptabl e phenological state isapproache d but whenclose ,th efemal e suddenly changes direction andflutter s to another one. Sometimes, aflowerhea d is inspected more intensely byflyin g several loops around it,sometime s thefemale swoul d alight and inspect itwit h her antennae orth e abdominal tip.Al lthi s time a rejection isstil l possible and usually severalflowerhead s are rejected before asuitabl e one has been found to proceed with egg-laying.Thi s behaviour isver y similar in Maculinea nausithous and M.teleius. The acceptable phenological state ofth e flowerhead whenfinall y depositing anegg ,i sdifferen t between the species (Thomas 1984a, Figurny &Woyciechowsk i 1998). It is important for the oviposition decisions ofth efemales . However, nextt othis ,th e complicated life cycle of both butterfly species suggest that the presence of host ants iso f equal importance.

In many occupied habitats the host plants are abundant while the host ant is relatively rare,s otha t most host plants are infac t unsuitable for oviposition duet o alac k of ants inthei r close vicinity. Survival ofth e progeny is initially dependent onth e availability of host ants' nests inth e close proximity of the host plant.Therefore , itwoul d givefemale s a comparative selective advantage andfitnes s increase, ifthe y coulddetec t hostant s soa s todeposi t eggs ontrul y suitable Sanguisorba plants only. Forth e source population atth e Kostrza site, pooling all datafo r thewhol e flight period,w e foundtha t Maculinea nausithousfemale s aggregate and oviposit in places where nests ofthei r host ant, Myrmica rubraar e present (figure 8.1). Neither the density of Sanguisorba officinalisplants , north e number offlowerhea d in budo r inflowe r appeared to helpt o explain the oviposition density. Maculineateleius female s oviposit independently ofth edensit y ofth e nests ofth e host ant Myrmicascabrinodis. Theovipositio n density was explained byth e number of host plants and the number offlowerhead s in bud.Mos t ovipositions were detected on patcheswit h lowvegetation .Thes e differences leadt oth e hypothesis that Maculinea nausithous can detect its host ant, while Maculineateleius react s to vegetation characteristics (Wynhoff 2001, chapter4) . The hypotheses mentioned abovewer e further tested ina n insectory experiment,wher e females of both species were free tochoos e between host plants on plotswit h Myrmicarubra o r Myrmicascabrinodis, orwithou t ants.W e initially observed the ovipositing females.Tw oweek s later, all flowerheads were enclosed insmal l netting sacst o capture the emerging caterpillars. Oviposition and caterpillar captures showtha t Maculinea teleius

163 Chapter 8: Synthesis

acid

s—-vSden Sbud^^-**^ Sflo

hum ' OM 1^^^^Ssca Wkpro^^^^ \\ V ^^^ Mtel high en tant. \ \ vR \ \ \vsrub diff\ \\

Mnau —\rub

Figure 8.1: Resulto fPC A analysis of oviposition observations ofMaculinea nausithous (=ovin) and M. teleius (=ovit). Explanatory variables are: mean number ofM. nausithous (=Mnau) and M. teleius (=Mtel) butterflies, number ofMyrmica rubra (=rub) and Myrmica scabrinodis (=sca) nests, total number ofMyrmica nests (=tant), number of Myrmica rubra (=srub) and Myrmica scabrinodis (=ssca) workers, number ofSanguisorba officinalis plants (=Sden), Sanguisorba flowerheads in bud (=Sbud), Sanguisorba flowerheadsi n bloom (Sflo), heighto fSanguisorba plants (=high), height difference between Sanguisorba and sward (=diff), Ellenberg value for humidity (=hum), acidity (=acid) and productivity (=pro) and the percentage ofbar e soil (=open).

selects hostplants on plotswit h Myrmica. Maculinea nausithousrespond s to vegetation structure andflowerhea d phenology characteristics, because the deveploment ofth eoffere d hostplants was very early. For both butterfly species the presence ofthei r hostan t species isimportan t in selecting flowerheads for oviposition.Thi s effect isclea r for thefirs t ovipositions while later phenological characteristics of theflowerhead s may become more important. Thus,th e results ofth e experiment provide further support for the presence ofth e host antspecie s having an impact onth e oviposition behaviour ofth efemales . However, they failt ogiv e indisputable evidence.

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Habitatselection of Maculinea teleius andMaculinea nausithous

Maculinea teleiusan d M.nausithous that parasitize as caterpillar inth e nests ofth e antspecie s Myrmica scabrinodis and M.rubra respectively , have narrowly defined habitat requirements (see chapter 1).Therefore , onewoul d expect that individuals areabl et o selectfo r sites that meet their requirements. Both butterfly species occur inhabitat s where their initial larval resource,th e host plant Sanguisorba officinalis, isabundan twhil e the ant nests are incompariso n less abundant. Fromthi s unequal occurrence in resources and because the home range ofth e Myrmica colonies isonl y limited, itfollow s that apar t ofth e host plant population represents asin k whenselecte d for oviposition. Inth e caseo f Maculinea butterflies,onl y host plants within the home range of a hostant s nest represent atru e resource, while host plants out ofth e home rangear e sinks.Afte r all, inth e proximity of a host plant being a resource,th e caterpillar hasa chanc e of beingfoun d by its host antspecies ,whil e ina sin k this Myrmica species is not present and therefore thecaterpilla r willdie .O na selectio n of moretha n60 0 plots inan d around Moerputten nature reserve,th e impact of presence or absence of host ant nests,vegetatio n composition,vegetatio n structure and microclimate onth e oviposition offemale s and onth edistributio n ofth e population was studied. Itappear s thatfemale s of both species prefer todeposi t eggso n host plants inth e close vicinity of ant nests. Furthermore, adults also select for plotswit h host ants. Plotswit h goodvegetatio n characteristics but lack of antswer e sometimes occupied, but only inyear s with high butterfly densities. Compared toth e results ofth eovipositio n experiment (chapter 4), these data are much more convincing.The y show,tha t it isth e antstha t are largely determining habitat quality andtha t distribution patterns are a result ofa n active choice ofth efemale s rather than asiev e inth e survival ofth e final instar caterpillars beforethe y enter the hostan t nests. Hence,th e random oviposition hypothesis hast o be rejected infavou r ofth e ant-mediated oviposition hypothesis. Maculineabutterflies , at least Maculineateleius, M. nausithous and M.alcon, are ,lik e most parasites, ablet ofin dthei r host.A straight forward calculation withth e limited knowledge available onth e mortality ofth e larval instars shows thatth e success ofth e initial phaseo f the reintroduction islargel y attributed toth e ant-mediated oviposition.Addin g an extra mortality of 70% due to random oviposition onsink st oth e high mortality that the larval instars already experience,woul d have madea reintroduction almost impossible (chapter 5).

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ForMaculinea teleiusw e haveals o additional anecdotal evidence suggesting ant-mediated oviposition from field observations. Sometimes, though only rarely,female s were observedwhe n they left the meadow BW. Wefollowe d these females without apparently disturbing them. Insevera l cases thefemal e started thetypica l flight behaviour when looking for hostplants andwer e observed depositing anegg .Afte r oviposition,w e always searched for Myrmica ant nests inth e direct surroundings of the chosen plant.W e always found onewithi n adistanc e of 2 m. Unfortunately, these ovipositions took placeo n meadows withtraditiona l mowing inth e beginning ofAugust ,whe nth e caterpillars are stillo nth e hostplant andwil l be removed withth e hay.

The spring andth eearl y summer ofth e year200 0wer e very coldan d wet.Therefor e the Sanguisorba officinalisplant s were not in budwhe nth e flight period of Maculinea teleiusstarted .Th efirs tfemale s had mated but almost noflowerhead s were available todeposi t eggs. Duringtw oday sw e observedfiv e ovipositions onth e leaves and stems of Sanguisorba officinalis and Vicia sepium. With sugar baits nextt oth este mo fth e used host plantw e attracted Myrmicaant swithi n ashor t period oftime .Apparently , evenwhe n accepting unsuitable hostplants,th efemale s stillfavou r locations with their hostants . However,thi s behaviour isunambiguousl y statistically testable only inth e beginning ofth efligh t period,whe n inyear swit h normal weather conditions there isa noverabundanc e of hostplants available. Later when manyflowerhead s are already occupied with one egg,other sfurthe r away from ant nestswil l beaccepte d too (Van Dyck etal. 2000,Wynhof f efal. chapter 4). Thefemale s of Maculinea teleiusrathe r accept a lower adoption chance than leaving the meadow andfindin g host plantswit hMyrmica scabrinodis nests nearby. Thus,whe n alldat a overth ewhol e flight period are combined,fo r this species no correlation between oviposition events and presence of host ant nestswil l befound . Thiswa s indeedth e case inth e source population in Kostrza (chapter 4) and inth e Moerputten population (Wynhoff, unpublished datafro m 1993). However, our data also show,tha t vegetation characteristics are also an important aspect of habitat selection.Thus ,thos e hostan t nests,whic h are located invegetatio n that areavoide d or usedt oa lesser extent byth e butterflies, can escape parasitism (Hochberg efal. 1994) .Sinc e infestation of a Myrmica colony through Maculineapredatio n increases its risko f extinction,th e empty sites can be recolonized by nests in unfavourable vegetation.

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Inth e Moerputten nature reserve,th e microdistributiono f Myrmica rubra iscorrelate dwit hth e microdistribution of Maculinea nausithouseggs ,and , independently, thedistributio n ofth e adults ofth e same species.Agai n independently, thesam e has beenfoun dfo r Maculineateleius an d Myrmica scabrinodis, albeit only forth e beginning ofth efligh t period.So ,th ean t species isa nexclusiv e resource for both butterfly species respectively. The facttha t hostant s can bedetected , has beneficial consequences with respect to increasing the survival probability ofth e offspring (which Maculineateleius only partly makes useof) . Itals o opensth e discussion to whether the butterflies are ablet o reducethei r hostan t nest density so much that they may initiate their own localextinction .Apparently , onth eon e hand the persistence ofth e butterfly populationdepend so n localrefuge sfo rth e host ant species.Thes e can befoun d in habitatwithou t Sanguisorba officinalis and on locations where the host plants develop buds inth e acceptable phenological stage only after thefligh t periodo f the butterfly. On theothe r handth e amount of co-occurence ofth e Myrmicaspecie s influences local mortality.Th e more non-host Myrmicanest sther e arei nth e close proximity of the host plant,th e more caterpillars will beadopte d by them,thereb y decreasing the extent of invasion intoan t nests andth e mortality. Through inefficient feeding behaviour and scramble competition, high mortalities occur inovercrowde d ant nests (Thomas & Elmes 1998, Thomas &Wardla w 1992).Th e consequences ofth e spatial distribution of host and non-host Myrmica colonies have been studiedfo r Maculinea rebeli, anadvance d predator usingth e rare host plant Gentiana cruciata(Hochber g etal. 1992, 1994, Elmes etal. 1996,Clark e era/. 1997). However, for Maculineateleius and M.nausithous, the availability of host plants and host ants isdifferen t andth e behaviour ofth e butterflies isdifferent , too. Therefore, new predictions should beteste d ina differentl y parameterised model.

Dispersaland the establishment of a possible metapopulation

The persistence of many populations isgenerall y increasedwhe nthe y consist of asyste m of several local subpopulations which exchange individuals with each other. Itgenerate s advantages with respectt o environmental and demographic stochasticity and decreases the chance of genetic impoverishment (Hanski& Gilpin 1997, Hanski 1999).Th e functioning of such a metapopulation iso nth eon e hand dependent on landscape characteristics such asth e number of connected patches,th e

167 Chapter 8: Synthesis distances between them and how easythes e distances can beovercome . Onth e other hand,specie s specific characters determinewhethe r therewil l beexchang e between subpopulations and how intense this exchange willbe . Within thesam e set of plots usedt o study the habitat selection of Maculinea nausithous and M.teleius afte r their reintroduction inth e new environment, we investigated their colonisation and abandonment pattern.Whil e inth e classical metapopulationapproach ,dynamic s of local populations and patterns ofexchang e between spatially disjoint patches of uniformly suitable habitat arestudied ,w e decided to concentrate ona fine r spatial level and study the same patterns on heterogeneous habitatwithi n one patchan d between patches aswell . Althoughth e individuals of bothbutterfl y species canselec t sitestha t contain boththei r resources,dispersa l leading to colonisation of suitable sites ata larger spatial scale seems to beconstrained .Thi s could because d by thefac t that the habitat quality ofth e unoccupied plots isto o low,o r because thedistanc e necessary to reachthes e plots isto o long. Effects ofth e degree of connectivity on colonisation and abandonment suggest that thesetw o processes are not random with respect toth e degree of connectivity ofth e plots. For both butterfly species, both habitat quality and connectivity affect colonisation patterns. Plotswit h high habitat quality are more likely to be colonised than plotswit h low habitat quality, however, with a lowdegre e of connectivity, their colonisation probability decreases. Dispersal constraints at individual level may affect habitat selection and have aneffec t onth e colonisation andabandonmen t rates inth e populations ofth etw oMaculinea butterfly species andthu s onthei r distribution.Maculinea nausithous colonizes unoccupied plots lessfrequentl y but covers langer distances. Maculinea teleius,however , shows only very small colonisation distances, which appear tosho w expansion - retraction processes within heterogeneous plots inon e particular habitat patch.Large r displacements arefoun d only very rarely inthi s species.Thes e datafi twel lwit h ourfinding s in mark- recapture studies (Wynhoff 1998b , chapter 3, chapter 6) andwit hth e published literature (Settele era/. 1996). Giventh e constrained dispersal andth e landscape inan daroun d Moerputten nature reserve,th e conservation of Maculinea teleiusshoul d concentrate on improving local habitat quality onth eoccupie d meadow and the meadows inth e closevicinity .A metapopulation willonl y beestablished , if highqualit y habitat patches and stepping stones at short distance from occupied siteswil l becreated .Conservatio n of Maculinea nausithous by creating aspatia l network of suitable patches seems to be likely to be more effective, sincethi s species can cover longer dispersal distances. Most likely

168 Chapter 8: Synthesis

informe r times,thes e processes of colonisation and abandonment of habitat patches took placequit e regularly andth e Maculinea populations were in dynamic equilibrium withthei r environment. In badtimes ,retractio n tocor e populations took place,fro m where empty patches could becolonize d after their recovery. However, nowadays Moerputten nature reserve represents only asmal l island of nature ina n intensively used agricultural landscape.I t isalmos t impossibletha t ametapopulatio n witha structur e needed by Maculineabutterflies , could existwithi n the borders of a nature reserve.Thi s iaonl y possible whenth e butterflies are giventh e possibility to spread into theagricultura l landscape. Clearly, sucha metapopulation would put new challenges to nature conservation.

Howdo they find the host ants?

Our results show that Maculinea females are ablet ofin d locations with their respective host ant species (chapter 4, chapter 5).W e canonl y speculate about the mechanisms which they uset od oso . Behavioural observations indicate that visual cues are important inth efirs t phaseo f searching aflowerhea d for oviposition.Onc e aMaculinea female hasfoun da flowerhead ina suitabl e phenological state,sh e relies onothe r cues.Fo r other butterfly species it has been showntha t chemical cues can bever y important to decide whether an eggwoul d be laid (Baur efal. 1998 , Honda ef al.1997) . Baylis and Pierce (1991) have showntha t females of theant - tended lycaenid species Jalmenusevagoras prefe r to lay egg batcheso n fertilized rather than unfertilized host plants.Th e importance ofvisua l cues is also shownfo rth e presence of ants (Jordano efal. 1992 ,Jordano ,pers . comm.). However, Maculinea teleiusan d Maculinea nausithous dono t search visually for ants. Mosto fth e rather small ant nests are subterranean and not between the roots or directly nextt o Sanguisorba officinalis plants. The oviposition behaviour does not include awal k alongth e stem ofth e host plant downwards to searchfo r them. So, ifth e presence of ants plays a role for depositing eggs, it islikel y that chemical cuesar e involved.Fo rth e Maculinea females itcoul d beth e scent ofth evolatil e pheromoneso rth e odour ofthei r Myrmica hostan t nests. Pheromones are important for the intra-an d interspecific communication of ants.Althoug h in most cases contact-chemoreception is very important, chemoreceptionove r longer distances byvolatile s has been shownt ooccu r frequently (Holldobler &Wilso n 1990). For Myrmica rubraan d Myrmicascabrinodis a number ofvolatil e and non-volatile pheromones from

169 Chapter 8: Synthesis

the Dufour gland have been identified andthei r function in interspecific communication has been partly clarified (Cammaertsetal. 1978,1981) . While thever y volatile compounds are notspecies-specific , the less volatile compounds usedfo r marking ofterritorie s are. Itwoul d bever y interesting to testth e behaviour ofgravi d Maculinea females when being confronted with these odours.Myrmica rubraworker s occasionally visit higher partso ftree s and herbswhe n searching for aphids. Duringthes e visitsthe y could leave trails of non-volatile pheromones onth eflowerhead s of Sanguisorba officinalis, which could bedetecte d byth e Maculinea females. Fromou rfiel d work,w e haveth e impression that this happens so rarely that it cannot explain theobserve d phenomena. It is more likely that one ofth e volatile components ofth e pheromones or other chemicals relatedt o nestodour s are involved inth eovipositio n behaviour of Maculinea butterflies.

Genetics ofreintroducing Maculinea butterflies

Genotype

After the reintroduction of Maculinea teleiusan d Maculinea nausithous, bothth e demography ofth efounde r populations aswel l asth e genetic consequences ofth e bottleneck were studied.Populatio n numbers increased instantly and loss of genetic variationwa s only mild.Th e low levels of genetic variation ofth esourc e populations did not have a negative impact onth e settling and increase ofth efounde r populations.Apparentl y the number of translocated founder individuals andth equalit y ofth e release site havea higher impact onsucces s thanth e genetic constitution ofth efounders . Hence,als owit h respect to genetic heterogeneity, no bottleneck effects could bedetecte d (chapter 7). In both Maculinea species,onl y atth eAco- K locus significant differentiation between source andfounde r population wasfound .Takin g into account thatth efligh t periods shiftedforward s andth e mean minimal life time ofth e adults has decreased,w ecom et oth econclusio n that the founder populations have experienced selection duet oth e changed ecological conditions atth efounde r site.

Bothfounde r populations have asignifican t negative F,svalu e (=inbreeding coefficient), which meanstha tthe y may insom ewa y beabl et o maintain higher levels of heterozygosity thanwoul d beexpecte d ina random

170 Chapter 8: Synthesis

mating population. Further research is necessary toelucidat e the processes responsible forthi s phenomenon. Still,wit h low levels ofgeneti c variation,th e chance of loosing variationthroug h stochastic events is high.Therefor e it is important to create metapopulationso f reintroduced speciesa tth e release sitet o compensate for local stochastic extinction of subpopulations.

Onth eothe r hand alo w level of heterozygosity in itself cannot always beconsidere d a problemfo r theviabilit y of populations orfo r thefitnes s of individuals, asther e are many rare and common species lacking ahig h level of heterozygosity, sucha sth e northern elephant seal (Mirounga angustirostris), theSout hAfrica n subspecies ofth e cheetah (Acinonyx jubatus) orth e Florida tree snail (Liguus fasciatus),a s anexampl e of anon - vertebrate species. In many cases it has been showntha t thiswa sth e result of historic bottlenecks, but it isno t necessarily the causefo r recent bottleneck events (Avise 1994). Other rareo r endangered species have been shownt o have normal or high levels of heterozygosity, such asth eflightles s parrot (Strigopshabroptilus) andth e Przewalski's horse (Equusprzewalski). So ingeneral ,a prediction onsurviva l of bottlenecked populations merely based onthei r genetical variation is not possible (see alsoVa n Hooft 2001). Dunham etal. (1999 ) suggest that it is likely that species with limitedwithi n population variability, or non-equilibrium population structure may bewel l adapted to persist inth efac e of extreme isolation or small population size. This may beth e casefo r species that inhabit naturally isolated,bu t relatively stable habitats. Obligate ant parasite butterfly species,whic h are adaptedt o specific habitat conditions,suc ha s M.teleius livin g in Myrmica scabrinodis nestso nwe t meadows, may have such adaptations aswell . The example of the reintroduction of the beaver to Sweden shows that aspecie s with low genetic variation can survive and even spread sucessfully over many years and generations (Ellegrenetal. 1993).Thi s case ofth e/Wacu/Znea-butterflie s alsodemonstrate s through the increase in population size,a balanced sex- ratioan dth e low chance of loosing heterozygosity inth efounde r population, thatth e Kostrza populations were good source populations for a reintroduction project. Obviously iti s importantt o enable agoo dstar to fa reintroduction project byfindin g areleas e sitewhic hfullfill s all ecological demands of aspecie s and by releasing manyfounde r individuals. However, it isobviou s that both species, butespeciall y M.teleius, hav e a very low mean minimal lifetime .Thi s makesthe m more susceptible to stochastic catastrophes.

171 Chapter 8: Synthesis

Phenotype

It is not only the bandsfro m the allozymes,whe nthe y appear during theexcitin g moments after the staining agar has been pouredou t over the white cellulose acetate plates that provide genetic information.Th e spots on thewing s being cutfro m the bodies before homogenizing themfo r the electrophoresis, willtel lthei r ownstory ,too .Size ,shap e and number ofth e spots can be measured and compared between populations to reveal relationships andt ofin d out,whethe r a possible bottleneck through reintroduction has becomevisibl e indifferen t wing phenotypes. Spot pattern variation has beenshow nt o havea high heritability insevera l butterfly species (Brakefield 1989). Furthermore, indications forth e biogeographic origin in historical times can bededuce d (Taberlet 1998). The Pleistocene period (2,400,000 to 13,000year s BP)consiste d of cyclic alternations dominated by major ice ages and short warm interglacials (Taberlet 1998, Hewitt 1996, Roy ef a/. 1996). Northern Europe asfa r as 52° N parallel,includin g mosto f Britain,wa s covered byth e Scandinavian ice sheet. The major mountain ranges had large ice sheets,too ,whil e between them the plains of Europewer e tundra,stepp e tundra and cold steppeo n permanently frozen soil. Flora andfaun a retreated from the coldt o refuge areas inth e south,fo r example Italy,th e Balcans or Turkey, and recolonised Central and Northern Europeagai n inth ewarme r periods.Whil e some species withdrew toon e refuge area,other s were split up into several refuges and evolved indifferen t ways during the periodo f separation. Later, through landscape barriers, postglacial spread couldtak e place along different dispersal routes.Throug h the long periods of separation conditions were created that allow theevolutio n of subspecies under influence of different selection through different environmental conditions. Furthermore, rapid longdistanc e (leptocurtic) dispersal will involve population bottlenecks and therefore losso falleles . It meanstha t populations atth e edgeo fth e rangewil lten d to be more homozygous comparedt othos e inth e centreo r closer toth e refuges. Inconclusion ,durin g the climatic oscillations ofth e Pleistocene,condition s were favourable for the evolution of divergence in species athighe r spatial scale,whil e at lower spatial scale the losso f heterogeneity can beexpecte d (Taberlet 1998, Hewitt 1996,Avis e 1994). Phenotypic andgenotypi c differences between populations canb e interpreted insuc h biogeographical context.A study onwin g patterns enables ust o include more individuals and more populations, since it canb e donewithou t killing butterflies. Moreover, we could include historic material

172 Chapter 8: Synthesis

from butterfly collections. For this part ofth e study,w e Jsl*- usedwing s of Maculinea teleiusfro m thesourc e population in Kostrza siKv••£•• (Poland),th e reintroduced population 1 . Iv*lMH inth e Moerputten nature reserve ^Hfilk^Sfif•K* t'"v. w-• (The Netherlands) and a population 'r>.att' vnn^Br»i ^^HflNUlK3dEnJ|• inth eWesterwal d region(Germany) . The latter isgeographicall y closest to •. y * 8^*-' the Netherlands. From other small and protected populations of Figure 8.2: Wing pattern of Maculinea Maculineabutterflies , samples were teleius, with measured spots associated by their number. collected by making slideso f anaesthetised individuals.Th e butterflies were putt o sleep one byon ewit h carbon dioxide to makea picture.Withi n 15minute s they recovered again.W etoo k photos inth e population ofth e Maraisd e Lavours (France) and additionally fromal l populations usedfo r the allozyme study. Furthermore we used cabinet specimen and all Maculineateleius butterflie s ofth e National Collection of the Museumfo r Natural History Naturalis at Leiden.Thes e include the historical Dutch populations in Helvoirt (which can beconsidere d apar to fth e old Moerputten population),th e riverine meadows of Roer and Swalm, several butterflies from theVosge s (F)an dth e extinct Belgian populations of Vilvoorde/Epeghem. Digitised images ofth ewings ,th e cabinet specimen andth eslide s were produced.Then ,w e measured area,perimete r and distance between numbered spots onth e left hindwing (figure 8.2). We recorded how many spots number 8wer e present (0-2)an d how many spotsw e couldfin d onth e leftfor ewing . We measured 336 malesan d25 1females ,o fwhic hw e used 256 males and194female s for a Principal Component Analysis (PCA).Alas , the measurements from the slides appeared todeviat e too muchfro m the others andwer e therefore excluded.Considerabl e phenotypic heterogeneity within andbetwee n populations wasfoun dwit hth eonl y exceptiono fthos e with aver y small sample size (Chindrieux, Vosges).

173 Chapter 8: Synthesis

te- Males 8 - B 6 • ^^m _4

Figure 8.3:Win g pattern of male Maculinea teleius, expressed in PCA coordinates. Abbreviations denote the populations (B=Vilvoorde (B), C=Chindrieux (F), KO= Kostrza source population (PL), MP= reintroduced Moerputten population (NL), NL=historical Dutch populations, V=Vosges (F), WW=Westerwald (D)).

Populations differed from each other inspo t size,win g size and spottiness. The largest butterflies withth e most complete wing pattern werefoun d in Chindrieux in France.Th eextinc t populations inth e Netherlands also consisted of large individuals with many spots. Conversely, butterflies from the source populations at Kostrza had comparably smaller wings. Spotswer e smaller, too,an d spots nr. 1an d 9an dt o a lesser extent 8an d 3wer e missing.Thes e differences were consistent in malesan dfemales . For both sexes,th efirs t four PCAaxe swer e significant inexplainin g the variationo f thewin g pattern measurements (figures 8.3 and8.4) . Then, populations were grouped together intoa n Eastern anda Western group,wit hth e populations from Kostrza, Moerputten and Westerwald together inth e Eastern group and the old Dutch,Belgia n and Vosges populations intoth eWester n groep.Withi n one group, no significant differences in PCAaxi s 1coordinate s could befoun d (West(¥): F=1.74, n=86, p=0.165, East(?): F=1.64,n=100 ,p=0.171 ,West(C) : F=2.77,n=132 , p=0.03, East(cf): F=2.15, n=119, p=0.098) with exception ofth e males from theWester n group.Thi s was duet oth e populationfro m theVosges ,whic h also hada ver y small sample size of only4 . Inconclusion ,mos t ofth e

174 Chapter 8: Synthesis

6- Females c \ 5 •

4 • 3 • xy'W v u__ i T MP * . .-.,-.^V / A. ... KO S 5 -10 • M . i) 15 V-\ NL -3 - -4 - • WW

Figure 8.4:Win g pattern of female Maculinea teleius, expressed in PCA coordinates. Abbreviations denote the populations (B=Vilvoorde (B), C=Chindrieux (F), KO= Kostrza source population (PL), MP= reintroduced Moerputten population (NL), NL=historical Dutch populations, V=Vosges (F), WW=Westerwald (D)). variation isdu et oth edifferenc e betweenth eWester n andth e Eastern group.Th e reasonfo rthi s phenomenon can befoun d in postulated differences in biogeographical origin sinceth e iceages . Iti s likely that bothgroup soriginat efro mdifferen t iceag e refugia (figure 8.4). Maculineateleius occurs only north ofth e Pyrenees. Mountain populations inZwitserlan d andAustri a are also located relatively low,no t exceeding 1600m (SB N 1987). Clearly, mountain ranges are effective distribution barriers tothi s butterfly. TheWester n group could have survived cold periods west ofth eAlp s ina refuge area,whic hwa s centered inth e Rhone delta.Du et oth e lower sea level,th e delta ofth e Rhone reached muchfurthe r intoth e Mediterranean than itdoe s now (Kurten 1972).Thi s refuge area (W) might also have hadextension s toth e regions ofth e tundra atth e northern border ofth eAlps .Accordin g to Lukhtanov & Lukhtanov (1994), Maculinea species occur on riverine meadows inth etaiga-zone . Such meadows could have existed along meltingwate r streams fromth e Alpine glaciers.Th esummer s inth e late Pleistocene steppe tundra vegetation werewarme r than inth e recent time tundra vegetation,an d open graslands including Sanguisorba officinalis, usedt o exist insuc h vegetation (Gamble 1999,Prin s 1997, Kurten 1972).Schmit t &Seit z (in press) found

175 Chapter 8: Synthesis

Figure 8.5: Putative location of refuge areas during last glaciation and later migration routes of Maculinea teleius. Broad line: southern border of Scandinavian ice sheet, dotted: glaciers, hatched:refug e areas:W =Wester n refuge, E1 = Eastern refuge 1, location of Eastern refuge 2 is unknown, arrows:colonisatio n routes.

strong indications forth eexistenc e ofCentra l European refugia for the butterfly species Erebiamedusa, an d it is likely,tha t Maculinea teleius survivedth e last iceag e ina simila r region.Wit h climatewarming , populations spread to Central France,t oth e South,t o the Langres andvi a theVosge st o Belgium andTh e Netherlands. Itwa s also possible to invade intoth e northern valleys ofth eAlp s after the largeglacier s melted (figure 8.5). The Eastern group,consistin g ofth e population of Kostrza,th e Moerputten daughter population andth eWesterwal d population, is possibly from aCentra l Asian refuge area.Thei r dispersal routewoul d have been much longer (Hewitt 1996,Taberle t 1998),whic h would explain the losses in phenotypic characters compared toth e Western group,a swel l asth e losso f genotypic variation inth e populations inth e South of Poland compared to those in Russia (Figurny etal. 2000). However, ourow n data show higher levels of genotypic heterogeneity inth e Kostrza population.Whil e the Kostrza and Westerwald populations have differentiated significantly inallozym e

176 Chapter 8: Synthesis

variation, as shown bya significan t FST-value(F ST=0.136, Cl(95%):0.23 3t o 0.039), the Westerwald population of Maculinea teleius shows more allozyme polymorphism rather than less.Significan t population differentiation was found atthre e loci: MDH-2,PG Man dAco-K . Thus,a combinatio n of phenotypic and genotypic data points inth edirectio n of more refuge areas in the East (E1an d E2).A putative refuge (E1) could have been located in Rumania and Bulgaria (see also Kurten 1972),a swa s alsofoun dfo r the grasshopper Chorthippusparallelus (Coope r etal. 1995) ,an dth e butterfly Polyommatus coridon(Schmit t & Seitz (in press b). Fromtha tregion , Slovacia, Czechia, Poland and Central Germany upt oth e valley ofth e river Rhine could have been colonised byth e Maculineabutterflies .Also ,a n invasion of alpine regions could have been possible. Inth eAlps , migration routes might have met eachothe r and ahybri dzon e could have been formed (see also Taberlet 1998an d Hewitt 1999). However, the number of studied populations isto o lowt ogiv e evidencefo r any of our hypotheses except for the separation into at leasttw o lineages. Itwoul d be interesting to study populations in Rumania, Russia and Czechia totes t whether the assumption of (at least) twodifferen t refuges inth e East issupported .Sinc e theAlp s could have been colonised byth e Western orth e Eastern lineage or both, populations there also deserve further study. Finally, itseem s that both lineages almost met each other atth e river Rhine in Germany. A similar phenomenon has beenfoun dfo r thewhit e stork Ciconia ciconia, where an apparently panmictic population exists oftw o lineages which meet inth e North of The Netherlands and Germany (Bairlein 2001). Ifthi s istrue ,the n the populations inth e Palatinate should resemble those of France,whil e the populations in Bavaria would be more likethos e in Poland.A s small and endangered populations would be involved insuc h astudy , iti s recommended to standardise the method of making slides from anaethetised butterflies, or usedigita lcameras . The results ofth ewin g pattern analysis of Maculinea teleiusd o not give evidence for abottlenec k duet oth e reintroduction.Th e reintroduced Moerputten population is not different from the Polishsourc e population nor in phenotype or ingenotype . Itals o does not show anytendenc y toshif t in wing patterns towards that ofth e extinct Dutch butterflies. However, itshows , that the butterflies from the historic populationfro m the reintroduction site resemble much morethos e from Belgium and France thanthos e fromth e Westerwald region.Th eWesterwal d populations are geographically closest toth e reintroduction site andwoul d normally have been thefirs t choice to serve assourc e population.W e decided to useth e much bigger Kostrza

177 Chapter 8: Synthesis

population,an ddoin g so,butterflie s from adifferen t biogeographical origin were translocated.Th efounde r population,bein gadapte d to climatic conditions inth e south of Poland, must have experienced considerable selection pressure. It is likely that this was expressed inth e shift of the flight period,th edecreas e in mean minimal lifetim e (Wynhoff 1998,chapte r 3)an d the significant differentiation between source andfounde r population atth e Aco-K locus (Wynhoff etal. 2001 , chapter 7). However, ecologically significant traits have most likely not been affected insuc ha wa ytha t itcoul d jeopardize thesucces s ofth e reintroduction. Nonetheless, ifanothe r extinct butterfly species or other animalwoul d be reintroduced to The Netherlands, it is recommended tofirs t investigate the genetics of (aserie s of possible) source populations and biogeographical origin of species to avoid excessive selection pressure.Th e Maculinea population might haveadapte d easier toth e reintroduction sitewhe na source population from Francewoul d have been selected.Whe nth e adults would have alonge r lifespan ,the y would probably beles s sensitive to environmental stochasticity.

Retaininggenetic diversity in a metapopulation ofMaculinea

The effective population size (Ne),whic h isa n important character ofa populationwit h respect to retaining genetic diversity, is reduced with fluctuations in population size, but it isals o affected byth e "architecture"o f the metapopulation (Gilpin 1991, Ray2001) . Subdivision into local interconnecting subpopulations, generally has a positive effect on persistence time,whe nth e extinction of subpopulations is independent from eachother . However, models predict thatth e more different the local populations are insize ,th e moreth e metapopulation resembles asingl e large population,whic h lowers persistence duet o an increasing effect of environmental stochasticity. It has been showntha t an uneven subdivision of a metapopulation reduces the effective population size moretha n expected (Ray 2001).Thi s meanstha t in metapopulations consisting of equal numbers of individuals,founde r effects are expected to happen morefrequentl y in systems of uneven unitstha n inthos e of equally sized units. Insuc ha situation two dispersing individuals are more likely to comefro m different populations.Whe n ametapopulatio n consists of aver y large and several very small populations,th efounder s are more likely to be bothfro m the large population. Ifth e large population goesextinct , only apar to f their genetic

178 Chapter 8: Synthesis

diversity will survive inth e small subpopulations since these have experienced abottlenec k in numbers and genetic drift (Ray 2001). Hence iti s important notonl y to establish metapopulations butals ot o manipulate their structure inorde r to preserve as much as possible geneticvariation .

Inth e case of Maculinea butterflies withthei r restricted dispersal,th e distance between subpopulations deserves particular attention. From mark- recapture studies we knowtha tfo r Maculinea nausithous, the exchange of individuals over 500 mwa s 8t o 10% i nyear s with high population densities (chapter 3,chapte r 7).Suc h high migration rates increase the persistence of subpopulations and avoidgeneti c drift, albeit also reducing genetic differentiation (Ray 2001). For Maculineateleius, distances between subpopulations most likely needt o beshorte rfo r the same rate ofexchange . Consequently, to increase population persistence and genetic diversity, several large connected subpopulations should beestablished . Basedo nou r experience,fo r Maculineanausithous th edistanc e betweenthe m should not be moretha n 1km ,whil e for Maculineateleius they should bewithi n 500m ofeac h other. Eventhoug h both butterfly species could be insensitive to genetic drift asa consequenc e of low population sizes, populations should be maintained as large as possible. The creation of such afin e scale metapopulationwithi n the highly fragmented and agriculturally used landscape, must bea seriou s problem for the reintroduced populations. Given the constraints indispersa l andth e highly stochastic negative effects of management of roadverge s and canal borders, it isuncertai nwhethe r the populations will beabl et o establish new populations bythemselves . The bestwa yt o ensure persistence ofth e resident populations by increasing the number of interconnected subpopulations withouttakin g the risko f anyfurthe r losswoul d then beth e application of anextende d reintroduction program. Genetic diversity can best beencourage d in metapopulations amonga network of habitat patches. However, such a networkwoul d soon surpass the borders of nature reserves and invadeth e landscape used byhumans . As habitat patches outside nature reserves are,a t least inTh e Netherlands, under controle of land managers and residentfarmers ,the y have substantial influence onth e persistence and genetic diversity of populations. Intheory , they can maintain populations by preventing deterioration of habitat and decline in numbers. Furthermore they can reduceth efrequenc y of human disturbance to local populations. Locally, onth e short term,thi s may bea s important for populations surviving onth e roadverge s between thefarm s as

179 Chapter 8: Synthesis

the management of nature reserves (see also Gibbs2001) . Forth e Burchell zebra in SouthAfrica ,th efirs t conservation program basedo ngeneti c information has been developed and implemented (Bowland era/. 2001). This program aimesa ta geneticall y based translocation scheme to retain genetic variability and avoid inbreeding rather than landscape architecure and management. For Maculinea butterflies under the given conditions, iti s recommended to also start asimila r program, butwit hth e integration of landscape management, since this isofte n the more effective wayt o tackle the problem ininsects .

Conservation ofMaculinea butterfliesat local level

The protection of Maculinea teleiuspopulation s should be concentrated on afavourabl e management ofth e host ant population, because the population dynamics ofth e butterfly isdependin g onth e density and spatial pattern of its host ant (chapter 4, 5an d 6). Females oviposit on Sanguisorba plants closet o host ant nests,an dth e presence of Myrmica scabrinodis isa n essentialfacto r with regardt o habitat selection. Duet oth e limiteddispersal , the persistence of populations isa tth e moment probably moredependen t on conditions atth e patch leveltha no nth e metapopulationlevel .Thus , conservation measures shouldfirs t concentrate on improvement of habitat quality. Inaddition ,th e population size per site should bea s high as possible to buffer against stochastic extinction,sinc e small insect populations are ata higher risko f extinction duet o chance events than large populations (Thomas &Jone s 1993)an d small populations experience higher losses of genetic variation (Gilpin 1991). The nutrient poort o mesophilic meadow vegetation (Molinietalia with strong Junco-Molinion aspect and species rich Molinio-Arrhenateretea) represents the vegetation composition ofth e Maculineateleius habitat . This vegetation needs aregula r mowing regime of onetim e peryear . Ifth e hydrological conditions with seepage of base-rich soilwate r and a high waterlevel (but dry during summer!) persist and as longa sth e mowing regime is kept the same,th e meadow BWwit h this vegetationform sa relatively stable site. Forth e benefit of Myrmica scabrinodis, it isessentia l to always have parts ofth e meadow with anope nvegetation . Mowing of habitatswit h Maculineateleius shoul d bedon e before the middle of Juneo r after the middle of September. Inth efirs t case there issufficien t regrowth of Sanguisorba officinalisbefor e the adults appear. Inth esecon d case,th e

180 Chapter 8: Synthesis

caterpillars are inth e ant nests before mowing and removing hay (Schurian 1984,Wynhof f 1992,Thoma s 1995). Inth e nature reserve, both Myrmica scabrinodis and Maculinea teleiuswil lthe n have a relatively safefuture ,a s long asthe y are restrictedt oth e meadow BW,eve nthoug hth e butterfly species isver y sensitive tostochasti c negative influence. Inth e nature reserve many more patches are availablewit h high habitat quality and presence of Myrmica scabrinodisnests .Occasionall y butterflies reachthes e patches andoviposition s have also been observed. However, mowing the vegetation inth e beginning of August preempts successful colonisation. Sinceth e establishment ofa metapopulations is needed,mowin g shouldb e shifted forward toth e beginning of June or latert o September. Inthi s wayth e suitable habitat of Maculinea teleiuswoul d increase substantially, which would increase their longter m persistence. Iti sals o recommended that comparably large areas of habitat should be managed to promote a similar habitatt o ensure ahig h carrying capacity with respect toth e Myrmica ants (Clarke efa/ . 1997). Nature conservation organisations should begive nth e support andth efunding s to perform such afin e scale, but quite expensive management. Duet oth ever y low mobility of Maculinea teleius, further expansion outside the nature reserve is nott o beexpecte d inth e present situation inth e nearfuture .Observation s of long distance dispersal are very rare. Forth e establishment ofa metapopulationa fin e grained networko fsteppin g stones with hostplants and host ants connecting habitat patches is needed,whic h is difficult to realize on roadverges . Ifanothe r network of populations of Maculineateleius shoul d beestablishe d atanothe r location,th e only possible way tod os owoul d be by reintroduction.

Inth e Moerputten nature reserve, Maculineanausithous i sa specie s of relatively transient successional habitats of abandoned meadowvegetation . The host ant Myrmicarubra doe s not occur inth e species rich Molinietalia meadows, butfind s its highest densities whenthes e vegetations are abandoned.Th etal l herbvegetatio n encompasses highest ant nest densities and is usedfo r searching byegg-layin gfemales . Forth e benefit ofMyrmica rubrai t isessentia lt o always have partswit h roughvegetatio n withina mosaic of different vegetation succession stages of abandonment. A 3- 5 yearturnove r of mowing is proposed toguarante e ahig hdiversit y of uncultivated grassland inon e habitat. Thisca n beachieve d by rotational mowing. However, it isdifficul t to keepsuc h atyp e of habitat suitable within the limitations of management plans of nature reserves. Duet oth e dynamics

181 Chapter 8: Synthesis

ofthi styp eo ftal l herbvegetatio n andth e relative mobilityo fth eadults , Maculinea nausithouswoul d also profit substantially from ametapopulatio n system of habitat patches inth e nature reserve and onth e roadverges .A s this butterfly occurs in patches of a highly dynamic nature,it s persistence is much more dependent onth e persistence ofth e metapopulation asa whole , rathertha no nth e persistence ofa singl e occupied habitatpatch .

Incontras t to Maculineateleius, Maculinea nausithousi sabl et o persist on small habitat patches. Dispersing adults usecorridors ,an d attention should begive nt oth e creation and management of connecting elements in the landscape.Th e management of roadverge s and stream borders should bedon e inth e samewa y asth e management of meadows. Inaddition ,th e population size per siteshoul d bea s high as possible to buffer against various kinds of (environmental, genetical or demographical) stochasticity.

Conservation of Maculinea butterflies at regional level

It might beclea r that reintroducing two parasitic Maculineabutterfl y species into The Netherlands appears to be risky andexceed s merely realisation ofth e ecological requirements ofth e species. Both butterflies and their host ants depend on highqualit y habitat kept bylo w intensity agricultural management. However, they are released intoa nature reserve, which isonl y asmal l part ina landscape, dominated byan dforme d withth e purpose of high intensity agriculture.Whe naimin g atth e long term persistence ofth e newlyfounde d populations, the adults havet o leaveth e nature reserve andfin dthei rwa y on verges among cornfield s and pastures. The expansion toth e road verges south of Moerputten has proven,tha t high quality patches are available.Th eadult s are ablet odetec t them and use these landscape elementsfo r further dispersal.Thus , habitat quality ofth e roadverges , ditch sides and embankments aroundth e nature reserveo f Moerputten arever y important.The yfulfi l arol ea sdispera l corridor but also as atemporar y or semi-permanent habitat (Oostermeijer &Wynhof f 1996). Buta tth e sametime ,sinc e Sanguisorba officinalis andth e Myrmicaant s are sensitive to management, the butterfly populations became very vulnerable to public management. To prevent problems andt o increase Myrmica ant nestdensit y andfacilitat efurthe r dispersalo fth e Maculineapopulations ,th e Province of Northern Brabant designated aMaculinea Action PlanAre a (Provincie Noord-Brabant 1997,figur e 8.6). Inthi s area the management of

182 Chapter 8: Synthesis

y. Bokhoven Hedikhuize/ 0 \ Engelen y Sompenen ( Zooislagen

^KiEngelenmeer Haarsteeg \ v/~ ~^\ Vlijmen^-^ / /Is Hertogenbosch \ Nieuwkuijk / /

Moerputten J ^^v Vlijmens Ven

( Vught )

Figure 8.6:Maculinea Action PlanAre a in the Province of Northern Brabant. Black circles: nature reserves, white circles: villages and cities, black line: Action Plan Area.

nature reserves and public property areas was adapted toth e ecological requirements ofth e Myrmica ant species. Hence,th evegetatio n inth e nature reserves ando nground s under public ownership, such as road verges,wer e managed ina wa ytha t encouraged initially acolonisatio n byth e hostan t species and later byth e Maculineabutterflies . Four actionswer eformulated : (a)adaptation s inth e management of nature reserves, (b)extension s of nature reserves, (c)adaptation s inth e management of, for instance,roa d verges, canal borders, ditch sides,embankments , and (d) monitoring of actions and effects. The action mentioned under (c) isessentia l for regional conservation.

183 Chapter 8: Synthesis

Adaptation inth e management means mainly that areas are mown less often. It iso fvita l importance that occupied Maculineahabita t isno t mowna t alldurin g the butterfly's flight period. Mowing only one side of road or ditch at a time reducesth edisturbanc e of mowing toth ewhol e insectfauna . Especially forth e benefit of Myrmica rubra,strip s ofvegetatio n were left unmownfo r oneo rtw o years. Furthermore, roughvegetatio n with flowers was mownonl y late inth e year. Participants inth e agreement ofth e Maculinea Action Plan are (a)th e Municipalities of Heusden, 's Hertogenbosch andVught , (b) State Forestry and "Natuurmonumenten", (c) Polder (Waterschap) de Maaskant, (d) Province of Northern Brabant, (e) Ministry ofAgriculture , Nature Conservation and Fisheries, Department South,(f ) Dutch Butterfly Conservation and (g)th e localfarmers .

Inth e designated Action PlanAre a (figure 8.5), 177plot swit h hostplants were searched for antsfo r a period of 15minute s to obtain relative density measurements. Thefirs t inventory was done in 1995 (Oostermeijer &Wynhof f 1996, Wynhoff etal. 2000). In 1997,th e management changed according toth e Maculinea Action Planan d in200 0 the antswer e counted againt o check whether there were effects ofth e changes in mowing regime (Wynhoff &Jansse n 2000). Thefirs t investigation of the antfaun a inth e Maculinea Action PlanAre a hasshown ,tha tLasius nigeri s byfa r the most common ant species.A negative correlation between the nest densities ofthi s species and Myrmicarubra (Spearma n r=-0.1772, p<0.05, n=177)an d Myrmicascabrinodis (Spearman r=-0.2345,p<0.005 , n=177)show stha tthes e species do not occurtogethe r but rather compete for habitat space (Oostermeijer &Wynhof f 1996,Wynhof f etal. 2000). Changes inth e management for the benefit ofth e Maculineabutterflie s should therefore increase abundance and nestdensitie s of Myrmica species (and Lasiusflavus) whil e Lasiusniger shoul ddecrease .

Sinceth e management ofth e road verges inth eActio n PlanAre a has beenchanged ,th ean tfaun a has indeedchange d (figure 8.7).Th e mowing frequency has been reduced which resulted in less disturbance. Ingeneral , significantly less plotswer e occupied byant s (Pearson x= 9-76, df=1, p<0.005) and on many plotsth e number of ant nests haddecrease d aswell . The pioneer species Lasiusniger i s now less abundant than before and nest densities are reduced,too .Th e number of plotswit h Myrmicaspecie s has not increased.Therefor e itseem s like they have not been ablet otak e over

184 Chapter 8: Synthesis

%60

50

40

30

20 10 Innil - L. niger L. flavus M.rubra M. M.ruginodis M. sabuleti scabrinodis

Figure 8.7: Changes in relative occupancy of plots with ants from 1996 (white bars) to 2000 (black bars) inth e Maculinea Action PlanAre a (n=180). Significant changes were found in Lasius niger (p<0.0001)an d Myrmica ruginodis (0.01

Ingeneral ,th e aimso f the adaptations inth e management of public

185 Chapter 8: Synthesis

propertywithi nth e MaculineaActio n PlanAre a have beenachieved .Excep t for two locations, adecreas e of Lasiusniger wa sfoun d incombinatio n with an increase in Myrmicaspecie s and Lasiusflavus. Through the reductions in mowingfrequency , more suitable sitesfo r Maculinea nausithous and Maculinea teleiusca n becreated .Th e more mobile M.nausithous ca n disperse betteran dfoun d newsubpopulation s omth e improved roadverges . ForM. teleiusw e expect positive effects only inth e longer term (Settele 1998). The great changes at Ruidigerdreef also show howvulnerabl e the implementation of anagreemen t such asth e Maculinea Action Plan is. Even though improvements in habitat quality can beachieve d byth e management, localaction swithi n the recentfligh t area ofth e Maculineabutterflie s can still havetremendou s effects onth e affected subpopulations. At the moment the butterfly metapopulationsyste m isstil lto o smallt o compensate for that. Therefore it isstil l necessary to putfurthe r effort intoth e information and motivation campaigns toal l participants ofth e agreement. Inaddition ,th e obligations signedfo r inth eagreemen t should becorroborate d byal l participants.

In conclusion.,

Thefiv e butterfly species ofth e genus Maculineahav e received major attention from European lepidopterists because ofthei r unusual life cycle as parasites of Myrmica ant nests and their declining status in most ofthei r range (chapter 2). The uninterrupted decline of these species has beena causet o develop aEuropea n Maculineaactio n plan to protect them inthei r total European range (Munguira & Martin 1999). Maculinea nausithous, Maculineateleius and Maculinea arionar e species mentioned inth e appendices IIan d IVo f the Habitat Directives.Thi sfac t puts extra impact on the needt ostrictl y protect populations andthei r habitats.An y actiont o protect apopulatio no f any Maculineawhereve r in Europe is therefore important and should besupporte d (Wynhoff & Munguira 2001).

Inth e European Maculinea Action Plan,reintroduction s are considered to be important inWester n Europe,especiall y when conservation of Maculineateleius i sconcerned .Specie s are notexpecte dt o readily adaptt o large scale landscape changes duet ofragmentatio n of habitat and climate warming,therefor e reintroduction programs are increasingly important. Our

186 Chapter 8: Synthesis

experiences can help inth e elucidation of problems of reintroducing highly specialised species and inth e conduction of reintroduction projects insuc ha waytha t the chance of success can be increased.A s aconsequenc e of landscape architecture andth ever y limited dispersal ofth e Maculinea species (and other comparable species with low mobility), itseem s that the establishment of a metapopulation isdifficult , if not impossible. Sincea metapopulation is required to realize persistence,founde r populations should bereintroduce d as metapopulationso r metapopulations should be established byadditiona l translocations. The reintroductions of Maculinea teleius and M.nausithous wer e conducted according toth e recommendations ofth e IUCN.Translocatin g a large number offounde r individuals appeared to be more important for the success thanth e genetic variation ofth e source population. Both Maculinea species seemt o be protected from losses through chance caused bylo w numbers of reproducing individuals, butthe y experienced selection underth e new environmental conditions atth e reintroduction site. However, since sufficient high quality habitat was available and adults were apparently able to select for such patches, populations could increase quite rapidly. Constraints indispersa l and deterioration of habitat through human interference are adange r toa longter m persistence.Whe n populations expand beyond the borders of nature reserves,thei r persistence is strongly linkedt o landuse.Th e conclusion must betha t reintroduction projects canb e successful. But however careful their scientific foundations have been,the y are pronet ofai lwhe n notaccompanie d byextension s and motivating campaigns aimed at local people, local authorities, nature conservationists and reservewarden s (figure 8.8).

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200 Watt WB, PA Carter & K Donahue 1986. Female choice of 'good genotypes' as mates is promoted by an insect mating system. Science 233: 1187-1190. Weeda EJ,R Westra , CWestr a &T Westr a 1987. Nederlandse Oecologische Flora. - Deel2 . IVN, VARA &VEWIN , De Lange/Van Leer, Deventer. Weidlich M & H Kretschmer 1995. Die gegenwartige Verbreitung des Schwarzblauen Ameisenblaulings (Maculinea nausithous [Bergstrasser 1779]) in Brandenburg. Naturschutz und Landschaftspflege in Brandenburg 4: 36-41. Weir BS 1996. Genetic Data Analysis 2. Second edition.Sinaure r Associates Inc., Sunderland Massachussetts. Weir BC & CC Cockerham 1984. Estimating F-statistics for the analysis of population structure. Evolution 38: 1358-1370. Wiens JA, NC Stenseth, BVa n Home & RA Ims 1993. Ecological mechanisms and landscape ecology. Oikos 66: 369-380. Winnemiller KO &A A Anderson 1997. Response of endangered desert fish populains to a constructed refuge. Restoration Ecology 5: 204-213. With KA &T O Crist 1995. Critical thresholds in species' response to landscape structure. Ecology 76:2446-2459 . Woyciechowski M 1991. Ginace motyle z rodzaju Maculinea i ich biologia. Pradnik. Prace Muzeum Szafera 3: 221-228. Wynhoff I 1991.Pimpernelblauwtje s weerteru g in Nederland? Vlinders 6(4): 18-22. Wynhoff I 1992. Herintroductie Pimpernelblauwtje (Maculinea teleius) en Donker Pimpemelblauwtje (M. nausithous) in Nederland 1990. Report Department of Nature Conservation, Wageningen Agricultural University, and Department of Nature, Forest, Landscape & Fauna, Minister/ of Agriculture, Nature Conservation & Fisheries, The Hague. Wynhoff I 1994. Hoe gaat het met Pimpernelblauwtje (Maculinea teleius) en Donker pimpernelblauwtje (M. nausithous)? Report Department of Terrestrial Ecology & Nature Conservation,Wageninge n Agricultural University and Minister/ ofAgriculture , Nature Conservation & Fisheries, The Hague. Wynhoff I 1996. Herintroductie Pimpernelblauwtje (Maculinea teleius) en Donker pimpernelblauwtje (M. nausithous) -vij fjaa r later. Report. Dutch Butterfly Conservation Wageningen and Ministry ofAgriculture , Nature Conservation & Fisheries, The Hague. Wynhoff 11997a. Data sheet Maculinea teleius. In: Helsdingen PJvan , LWillems e & MCD Speight (eds): Background information on invertebrates of the Habitats Directive and the Bern Convention. Part I- Crustacea, Coleoptera and Lepidoptera. Nature & Environment, No 79. Council of Europe Publishing, Strasbourg. Wynhoff 11997b. Data sheet Maculinea nausithous. In: Helsdingen PJvan , LWillems e & MCD Speight (eds): Background information on invertebrates of the Habitats Directive and the Bern Convention. Part I- Crustacea, Coleoptera and Lepidoptera. Nature & Environment, No 79. Council of Europe Publishing, Strasbourg. Wynhoff 11998 a. Lessons from the reintroduction of Maculinea teleius and M. nausithous in the Netherlands. Journal of Insect Conservation 2:47-57 . Wynhoff 11998b. The recent distribution of the European Maculinea species. Journal of Insect Conservation 2: 15-29. Wynhoff I, H deVos , MP van Zuijen, HHT Prins & PM Brakefield. 2001c. Genetic variation is retained after reintroduction of the butterflies Maculinea nausithous and Maculinea teleius. Submitted. Wynhoff I& S Janssen 2000. Meer mieren voor pimpernelblauwtjes. Report VS 2000.28, De Vlinderstichting, Dutch Butterfly Conservation, Wageningen. Wynhoff I& ML Munguira 2001e :Actio n Plan for Maculinea Butterflies in Europe. Proceedings of the Workshop "Protection of Endangered Large Blue Butterflies on Zovje wet Meadows". Ministry of Environmental Protection and Physical Planning, Republic of Croatia,Zagreb . Wynhoff I,JG B Oostermeijer, M Scheper & JG van der Made 1996. Effects of habitat fragmentation on the butterfly Maculinea alcon inth e Netherlands. In: Settele J,C Margules, P Poschlod & K Henle (eds.): Species Survival in Fragmented Landscapes. The GeoJournal Library vol.35 , Kluwer, Dordrecht, Boston, London, p. 15-23.

201 Wynhoff I, MGA van der Heijden,J G van der Made, S Plat, HHT Prins, Mva n Steenis &M Woyciechowski 2001a. Oviposition by Maculinea nausithous and Maculinea teleius butterflies in relation to their host plant and specific host ants.Submitted . Wynhoff I& JG Van der Made 1995. Reintroduction of Maculinea teleius and Maculinea nausithous inth e Netherlands in 1990. Proceedings of the Section Experimental & Applied Entomology NEV Amsterdam 6: 79-80. Wynhoff I, Fva n Langevelde, HOlff , M Grutters, PM Brakefield & HHT Prins 2001b. Looking for the ants: habitat selection of two Maculinea butterflies and their Myrmica host ants. Submitted. Wynhoff I& CAM van Swaay 1995. Bedreigde en kwetsbare dagvlinders in Nederland. Basisrapport met voorstel voor de Rode Lijst. (Threatened and vulnerable butterflies in The Netherlands, basic report and proposal for the Red List). Report Nr. VS95.11. De Vlinderstichting (Dutch Butterfly Conservation), Wageningen. Wynhoff I,CA M van Swaay, AMH Brunsting& JG van der Made (2001d): Conservation of Maculinea butterflies at landscape level. Proceedings of the Section Experimental & Applied Entomology, NEV Amsterdam 12: 135-141. Zuidhoff AC, JHJ Schaminee & Rva n 't Veer 1996.Molinio-Arrhenateretea. In: SchamineeJHJ , AHF Stortelder, EJWeeda , E Dijk, H Doing & SM Hennekens. De vegetatie van Nederland. Deel 3. Plantengemeenschappen van graslanden, zomen en droge heiden. Opulus Press, Uppsala, p. 163-226.

202 Summary/Samenvatting/Zusammenfassung

Summary

Thefiv e butterfly species ofth egenu s Maculineahav e received major attentionfro m European lepidopterists because ofthei r unusual life cycle as parasites in Myrmica host ant nests. In mosto fthei r range, Maculinea butterflies have shown a remarkable decrease in numbers of populations and individuals per population.Thi sdeclin e is mainly duet o agricultural improvement aswel l as abandonment, both resulting in marked changes in habitat. Furthermore many populations were lost duet o habitat destruction for houses, infrastructure,afforestatio n and landdrainage .Thi s resulted ina sever e decline ofthei r distribution ranges and an increaseo f fragmentation and isolation ofth e surviving populations.

Maculineateleius (scarc e large blue butterfly) and M. nausithous (dusky large blue butterfly) occur on mesophilet o nutrient poor moist meadows. For Maculinea teleius,habitat s are mown oncet otwic e a year, depending onthei r productivity, while Maculinea nausithous alsothrive s in taller herbvegetatio n that is mown every third tofift h year. InTh e Netherlands, both species usedt o occur mainly inth e riverine landscapes ofth e provinces Limburgan d Northern Brabant. Theywen t extinct inth e 1970s. In 1990 both Maculinea specieswer e reintroduced inth e nature reserve Moerputten in Northern Brabant, following the recommendations of the IUCN.Wit h aver y highdensit y of Sanguisorba officinalis andth e host ant species Myrmicarubra an d Myrmica scabrinodis present, this nature reserve wasfoun dt o beth e onlyfeasibl e reintroduction site.A sa source we usedth e metapopulations of both butterfly species nearth e village Kostrza inth e valley of the riverWisl a south of Krakow inPoland . These metapopulations consist of several thousands of adults peryea r andwoul d not beendangere d if a high number of individuals were taken away.

The reintroduced populations of Maculinea teleiusan d Maculinea nausithousbehave d differently after release.Th e population ofM. teleius expanded during thefirs t three years.Then ,afte r adecreas e in numbers duet o overexploitation ofth e host ant population,th e population size levelled at 300 adults per year. This happened twice,sinc e in 1995th e occupied meadow was accidentally mown inth efligh t period. Maculinea teleiusappeare d to show only restricted dispersal. Untilth e present day, the only occupied meadow isth e onewher e the butterflies were releasd in

203 Summary/Samenvatting/Zusammenfassung

1990.

The newly established population ofM. nausithous declined inth eyea r after the reintroduction butexpande d later. After several yearswit h very high population size,th e number of butterflies isagai n decreasing.Th e butterflies leftth e meadow where they were released and settled onth e railway embankment crossing the nature reserve.Thi s appeared to beth e sitewit hth e highest density of Myrmica rubranests ,th e specific hostan t species for this butterfly. Two years later, the roadverge s atth e street Ruidigerdreef atth esouther n side ofth e Moerputten nature reserve were colonized.Thi s second subpopulation also spread and increased in numbers.Thes e subpopulations were separated byabou t 500 mbu t exchanged 8t o 10%o f adults during thefligh t period.Agai ntw o years later, athir d subpopulation at adistanc e of moretha n 5k mwa sfounded . Thissubpopulatio n isstil lver ysmall .

Although the results are quite positive with respect toth e numberso f butterflies, somefeature s of potential concern have also beenfound .Bot h species, butespeciall y M.teleius, hav e aver y low mean minimal adult life span,whic h makesthe m moresusceptibl e to stochastic catastrophies. Furthermore, it has beenfoun d that thefligh t periods ofth e reintroduced populations differ from that ofth e source populations andfro m that ofth e historic Dutch populations. Maculineateleius i so nth ewin g during the month of July, Maculinea nausithousfro mth e middleo fJul y toth e middle ofAugust . Inth eforme r Dutch populations and in Kostrza,a normal flight periodo f bothspecie s endures untilth e endAugust .

Maculinea nausithous and Maculineateleius butterflie s are dependent ontw o sequential sources offood , namely the host plantSanguisorba officinalis andfo r each butterfly species adifferent , specific, Myrmicahos t ant.Th e eggsar edeposite d on Sanguisorba officinaliso fcertai n phenological stages differing between the butterfly species.Th e L1t oL 3 instar caterpillars feed onth e hostplant for twot othre e weeks.Then ,the y dropt oth e ground to be collected byworke r antso f Myrmica species.I n the ant neststhe y feed onan t grubs, hibernate and pupate. Maculinea nausithousparasitize s onth e nests of Myrmica rubra,whil eMaculinea teleius stays inth e nestso f Myrmica scabrinodis. Myrmica ants havea limited home range, normally not exceeding 2 maroun d the nests. Therefore, in most occupied habitats the host plants are abundant while the host ant is relatively rare,s otha t most host plants are infac t

204 Summary/Samenvatting/Zusammenfassung

unsuitable for oviposition due toa lack of ants inthei r closevicinity . It would givefemale s acomparativ e advantage ifthe y could detect hostant s so ast odeposi t eggs ontrul y suitable Sanguisorba plants only. However, Maculinea butterflies arethough t tooviposi t randomly with respect toth e occurence of host ants (=random oviposition hypothesis).Th e phenology of achose n flowerhead and vegetation characteristics arethough t to be the most important variables afemale s reacts uponwhe n depositing an egg. Weteste d oviposition in response toth e density of both the sources offoo d (=ant-mediated oviposition hypothesis) bycomparin g adjacent patches differing in host plant and hostan t nest density. This part ofth e reserach was performed atth e Kostrza site. Maculinea nausithous females appeared toaggregat e andoviposi t in placeswher e nestso fthei r host ant, Myrmicarubra ar e present. Maculineateleius female s oviposit independently ofth e density ofth e nests ofth e host ant Myrmica scabrinodisbu t rather respond to vegetation andflowerhea d characteristics. Basedo nthes e differences weformulate d the hypothesis that Maculinea nausithous can detect its host ant,whil e Maculinea teleius reacts to vegetation characteristics.

The idea of ant detection wasthe nfurthe r tested ina n insectory experiment. Females of both species were freet o choose between host plants on plotswit h Myrmicarubra o r Myrmicascabrinodis, orwithou t ants,whil e vegetation characteristics were similar between plots. First,w e took notes of oviposition observations, laterth eflowerhead s were encapsulated with small curtain bagst o capture the caterpillars. The oviposition observations show us howfemale s reactwhe n they can choose optimal sites ina overabundanc e of emptyflowerheads . The caterpillar captures include the choices whenth e bestflowerhead s are already occupied. For both Maculinea species, inth e beginning ofth e flight period the presence ofth e respective hostan t species hasa n influence onth e oviposition ofth e butterflies. ForMaculinea teleius, caterpillar captures also showtha t females prefer hostplants on plots with Myrmica.Maculinea nausithous later responds tovegetatio n structure and flowerhead phenology characteristics, because the development ofth e offered hostplantswa sver y early. Hence,firs t indications have beenfoun d that for both butterfly species the presence ofthei r host ant species is important inselectin g flowerheads for oviposition.Thi s effect isclea r for thefirs t ovipositions while inth e latefligh t period a heavier weighting of vegetation structure andflowerhea d phenology characters wasfound .

205 Summary/Samenvatting/Zusammenfassung

Giventh e narrowly defined habitat requirements of Maculinea teleius and Maculinea nausithoustha t livea s acaterpilla r inth e nests ofth e ant species Myrmica scabrinodis and M.rubra respectively , onewoul d expect that individuals areabl et o selectfo r sites that meetthei r requirements. In contrast toth e situation inth e insectory, inth efiel dthei r first larval resource,th e host plant Sanguisorba officinalis, isabundan t while thean t nests are incompariso n less abundant. Therefore, apar t ofth e host plant population represents asin kwhe n selected for oviposition due to the limited home range of the ant nests. Inth e case of Maculinea butterflies, the presence of host ants closet oth e host plants determines the suitability of the host plant as beinga tru e resource ora sink . Only inth e proximity of a host plant being aresource , hasth e caterpillar achanc e of being found by its hostan t species. Inth e proximity of asin k the requiredMyrmica species is not present andtherefor e the caterpillar will die.Th e impacto f presence or absence of host ant nestso nth e oviposition offemale s and onth e distribution ofth e population was studied ona selectio n of more than60 0 plots inan d around the Moerputten nature reserve.W e tested the oviposition of Maculinea nausithousb ycountin g empty egg shells on flowerheads that were collected in September. Sincethi s methodwa s not usefulfo r Maculinea teleius,w e observed females ofthi s species during thefirs t half of thefligh t period. Itappeare d that females of both species prefer to deposit eggs on host plants inth e close vicinity of ant nests. Then,th e habitat selection of the adultswa steste d at ahighe r spatial level byanalysin g the distribution since 1990o n plots.Th e vegetation composition,vegetatio n structure,an tfaun a and microclimate of these plotswa s described and measured indetail .Th e analysis clearly shows, that adults select for plotswit h host ants aswell . Only inyear s with high butterfly densities, plotswit h goodvegetatio n characteristics but lacko f antswher e occupied,too .Thes e data enable ust o rejectth e random oviposition hypothesis. Furthermore, it isargue d that the success ofth e reintroduction is largely attributed toth e ant-mediated oviposition.

It is puzzling that, although the adult individuals of both butterfly species can select sitestha t contain boththei r larval resources, dispersal leading to colonisation of suitable sites at alarge r spatial scale seemst o beconstrained . Itappear s that highqualit y plots are not easily colonized whenthe y arefa r awayfro m occupied plots. Incontrast , low quality plots are easily colonized whenthe y are closet o occupied high quality plots. Dispersal constraints at the individual level affect habitat selection and have aneffec t onth e colonisation and abandonment rates inth e

206 Summary/Samenvatting/Zusammenfassung

populations ofth etw o Maculineabutterfl y species and thus onthei r distribution.Maculinea nausithouscolonize s over longer distances than Maculinea teleius,bu t both species are limitedwit h respect to covering distances. For Maculinea teleiusth e pattern of colonisation and abandonment resembles een expansion-retraction pattern within the heterogenous plots onth e occupied meadow BW.Th e limited dispersal implies consequences for the populations.Th e meadow BWshoul d be kept at high quality for Myrmicascabrinodis and Maculineateleius. This butterfly can only reach adjacent meadowswhil e long distance dispersal is nott o beexpected . For Maculinea nausithousi t is recommended to establish a metapopulation. Itmean stha t changes inth e management in the nature reserve but also onverge s and canal borders inth e surrounding areneeded .

Fiveyear s after the reintroduction, ageneti c analysis of source and founder poulations was performed.Th e reintroduced populations of both butterfly species did not pass a bottleneck interm s of a low number of founder individuals. Population numbers increased very rapidly and losso f genetic variationwa s only mild.Th e source populations appeared to have only low levels ofgeneti c variation (inallozym eelectrophoresis ) butthi s did not have a negative impact onth e settling and increase ofth e founder populations.Apparentl y the number oftranslocate d founder individuals andth e quality ofth e release site have a higher impact on success than the genetic constitution ofth efounders .

In both Maculinea species only atth eAco- K locus significant differentiation between source andfounde r population wasfound . Taking intoaccoun t that thefligh t periods shifted forwards andth e mean minimal lifetim e ofth e adults hasdecreased ,w e comet oth e conclusion that the founder populations have experienced selection duet oth e changed ecological conditions atth efounde r site.Thi s conclusion issupporte d by ananalysi s ofth ewin g patterns ofth efounde r population,th e reintroduced population,th e historic Dutch population and additional ones in France, Belgium and Germany. The historic Dutch,th e Belgian andth e French population arealtogethe r phenotypically different fromPolish , German and Moerputten population.W e postulate that this isdu et o different Pleistocene refugia west and east ofth eAlps .Apparently , butterflies from adifferen t biogeographical originwer e translocated toa location outside their naturally colonized range.Th efounde r population, being adapted to climatic conditions inth e south of Poland,mus t have

207 Summary/Samenvatting/Zusammenfassung

experienced considerable selection pressure.

Bothfounde r populations have asignifican t negative FIS value,whic h meanstha t they are insom ewa y able to maintain higher levels of heterozygosity thanwoul d beexpecte d ina random mating population. Further research is necessary toelucidat e the processes responsiblefo r this phenomenon.

Fora longter m persistence, the reintroduced Maculineapopulation s needt o establish metapopulationst o protect themfro menvironmental , demographic and genetic stochasticity. Eventhoug h adults appeared to beabl et ofin dthei r host ants,thei r dispersal and colonisation ability was very restricted.Maculinea teleius ca nestablis h a metapopulationwithi n the borders ofth e nature reserve. Nextt o ensuring highqualit y habitat on the occupied meadow byconstan t management, adjacent meadows should be managed inth e samewa y so ast o enable colonisation by dispersing adults.Colonisation s of other nature reserves is nott o be expected without further reintroductions. Maculinea nausithous is dependent onth e road verges inth e surrounding ofth e nature reserve as habitatsfo r subpopulations of anetwork . Thus,thi s species isver y sensitive toth e management byorde r of official authorities andb y residents.A local Maculinea Action Plan hasshown ,tha t after changes in management, the antfaun a onth everge s has improved. However, atth e sametime , Maculineahabita t has beendestroye d by illegal private activities.Again ,colonisatio n of sites ata longer distance isonl y possible by meanso f reintroduction projects. Inconclusion ,constraint s indispersa l and deterioration of habitat through human interference are adange r to a longter m persistence ofth e Maculinea populastions.A s most nature reserves are small and embedded ina nagricultura l landscape, butterflies havet o leave nature reserves and spread between them.Thei r persistence is thenstrongly linkedt o landuse. Reintroduction projects, if performed carefully, can bea usefultoo l in nature conservation to compensate for large scale changes sucha s climate warming,an d they can be successful. But however careful their scientific foundations have been,the y are pronet ofai lwhe n not accompanied bythourough ,wel l used information and motivating campaigns aimed at local people,loca l authorities, nature conservationists and reservewardens .

208 Summary/Samenvatting/Zusammenfassung

Samenvatting

InEurop a komenvij f soorten blauwtjes van hetgenu s Maculinea voor. Door hun bijzonderelevenswijz e als parasieten ind eneste n vanMyrmica mieren hebbenzi j deaandach t van veelvlinderdeskundige n opzic h gevestigd. Helaasvertone n alle soorten binnen grote delenva n hun verspreidingsgebied een sterke achteruitgang inaantalle n populaties en ook in populatiegrootte. Deachteruitgan g wordtveroorzaak t door de modernisering ind e landbouw,waardoo rvee l percelen intensiever worden gebruikt enander eworde n verlaten. Inbeid e gevallen verandert het leefgebied van devlinder s insterk e mate. Daarnaastzij n leefgebieden verloren gegaan door deaanle g vanwoonwijke n en infrastuctuur, door de aanleg van productiebossen endoo rverdroging .Vee l van de resterende leefgebieden liggen inee nversnipper d landschap waardoor en isolatie van populaties plaatsvindt.

Hetpimpernelblauwtj e (Maculineateleius) e n hetdonke r pimpernelblauwtje (Maculinea nausithous) komenvoo r opmati g voedselrijke tot voedselarmevochtig e hooilanden enverlate n hooilanden. Het leefgebied van het pimpernelblauwtje moet, naar gelangd e productiviteit vand e bodem,ee nto ttwe e keerpe rjaa r gemaaidworden . Hetdonke r pimpernelblauwtje komtdaarnaas t voor in ruigerevegetatie ,

Het pimpernelblauwtje (links) en donker pimpernelblauwtje zijn in heel Europa bedreigd en vallen onder de habitat-richtlijn. Maculinea teleius (left)and M. nausithous are threatened all over Europe and are both Habitat-directive species.

209 Summary/Samenvatting/Zusammenfassung

die slechts omd edri e totvij fjaa r wordt gemaaid. InNederlan d kwamen beide soorten voor ind e rivier- en beekbegeleidende graslandenva n Limburge n Noord-Brabant. Zezij n ind ezeventige rjare n verdwenen uit Nederland.

In 1990werde n beide Maculinea soorten geherintroduceerd inhe t natuurgebied Moerputteni n Noord-Brabant (in het kader van het Beschermingsplan Dagvlinders van het Ministerie van Landbouw, Natuurbeheer enVisserij) ,volgen s devoorschrifte n van het IUCN.D e Moerputten bleek deenig evoo r herintroductie geeigende locatie,omda t dewaardplan t Sanguisorba officinalis(grot e pimpernel) inhog e dichtheden aanwezigwa s end ewaardmiere n Myrmica rubra(rod e steekmier) en Myrmica scabrinodis (ruwknoopmier) eveneens werden aangetroffen.Al s bronpopulaties fungeerden een aantal metelkaa r in verbinding staande populates van beide vlindersoortenrandom he t dorpje Kostrza ind evalle iva nd e rivier deWisl ate nzuide n van Krakow in Polen. Dezezogenaamd emetapopulatie s bestaan uitduizende nvlinder s perjaa r enzoude n geen schade ondervinden van hetweghale n vanee nflin k aantalvlinder s voor de herintroductie.

Nad e herintroductie ontwikkelden de nieuwe populaties van pimpernelblauwtje endonke r pimpernelblauwtje zich nogalverschillend . De populatie van het pimpernelblauwtje breidde zichd e eerste driejaa r behoorlijk uit. Daarna volgde eenflink e teruggang,veroorzaak t door te hoge belasting van de mierennesten,waarn a de populatie schommelde rond 300vlinder s perjaar . Dit proces herhaalde zich, nadat in 1995 het hooiland per ongeluk beginaugustu s werd gemaaid.He t pimpernelblauwtje bleek heel honkvast. Totvandaa g isallee n dat hooiland gekoloniseerd waarop devlinder s bij de herintroductie in 1990zij n vrij gelaten.

Hetdonke r pimpernelblauwtje liet nad e herintroductie eerst een teruggang inaanta l zien, voordat de nieuwe populatie begont egroeien . Naenkel ejare n met bijzonder veelvlinder s nemend eaantalle n nuwee r af. Op het hooiland waar zevri jzij n gelaten komenz e niet meervoor . Er vestigde zicheers t een populatie opd espoordij k die dwars door het natuurgebied loopt. Later bleek dat dit de plekwa s waar de hoogste nestdichtheid vand e specifieke waardmier Myrmicarubra voorkwam . Tweejaa r laterwerde n dewegberme nva n de Ruidigerdreef ten zuiden vand e Moerputten gekoloniseerd. Ookdez e subpopulatie namsne l in

210 Summary/Samenvatting/Zusammenfassung

omvangtoe . Uitvangst-terugvangs t onderzoek bleek dat 8-10%va nd e teruggevangen vlinders afkomstigwa s uitd e andere subpopulatie.Wee r tweejaa r laterwer d eenderd e subpopulatie opee nafstan d van ruim5 k m gevormd. Deze subpopulatie isechte r kleingebleven .

Wat deaantalle n vlinders betreft, lijken de resultaten vrij positief, maar erzij noo k enkele punten vanzorg .Beid e soorten,maa rvoora l het pimpernelblauwtje blekengemiddel dee n zeer korte levensduurtehebben . Hierdoor worden zij gevoelig voor onvoorspelbare tegenslagen.Verde r bleek datd evliegtij d van degeherintroduceerd e populaties verschilt van dieva nd e Poolse bronpopulaties. Ook devliegperiod e vand eoud e Nederlandse populaties was later. Het pimpernelblauwtje vliegt nuvrijwe l alleen ind e maandjuli , het donker pimpernelblauwtje vliegt van midden julito t midden augustus.Vroege r in Nederland vlogen en in Kostrza vliegen desoorte nto t eind augustus en soms nog later.

Het pimpernelblauwtje en hetdonke r pimpernelblauwtje zijn afhankelijk vantwe e voedselbronnen,t ewete n dewaardplan t Sanguisorba officinalis envoo r iedere vlindersoort een andere specifiekeMyrmica waardmierensoort. Deeitje s worden afgezet op Sanguisorba officinalis, waarbij desoorte n echter verschillende ontwikkelingsstadia prefereren.D e rupsen inhe t eerste totderd e stadium voeden zich met deze waardplant gedurende twee tot drieweken .Vervolgen s latenz e zicho pd e grand vallen enworde n door Myrmica werksters meegenomen naar het mierennest. Inhe t mierennest voeden zezic h verder van mierenlarven.Z e overwinteren enverpoppe n er. Hetdonke r pimpernelblauwtje parasiteert ind e nestenva n Myrmicarubra, terwij l het pimpernelblauwtje verblijft ind e nestenva n Myrmicascabrinodis. Dewerkster s hebben een klein foerageergebied en komen normaal nietverde r dantwe e meterva n hun nest. Invee l leefgebieden komtd ewaardplan t algemeene n inhog e dichtheden voorkomt,terwij l de mierennesten inverhoudin g zeldzaam zijn. Hierzij nvee lwaardplante n eigenlijk ongeschikt ome r eitjes opa ft e zetten, omdate r geen mierennesten dichtbij zijn.Vrouwtje s zouden daaromee nvoordee l hebben,al sz ewaardmier(nesten ) zouden kunnen detecteren,zoda t ze hun eitjes alleen afzetten opdaadwerkelij k geschikte waardplanten. Erword t echter van uitgegaan,da t Maculinea vrouwtjes huneitje s met betrekking tot de mierenwillekeuri g afzette n (=random oviposition hypothesis).D eontwikkelin g van de bloemhoofdjes end e

211 Summary/Samenvatting/Zusammenfassung

De Bijenwei (BW) in de Moerputten: leefgebied van het pimpernelblauwtje. De vlinders vinden er alles om te kunnen overleven: veel waardplanten en nectarplanten en zeer veel nesten van de ruwknoopmier Myrmica scabrinodis.

Themeadow BW (Bijenwei) in the Moerputten: home of Maculinea teleius.A high density of thehostplant, many nectar sources and many nests of thespecific hostant Myrmica scabrinodis can be met at this site.

vegetatiekenmerkente r plaatse zoudend e meest belangrijke eigenschappen zijn, waarop eeneiafzetten d vrouwtje reageert. Eri s getoetst of deeiafzettin g niettoc h een repons opzowe l bloemhoofd-al s mierennestdichtheidzo u kunnen zijn (=ant-mediatedoviposition). Di twer d gedaan door bij hetdorpj e Kostrza vlakken tevergelijken ,di e in waardplant- enwaardmiernestdichthei dverschilden . Het bleek datd e vrouwtjes van het donker pimpernelblauwtje zichvoornamelij k daar ophouden en eitjes afzetten,waa r veel nesten van hunwaardmie r Myrmicarubra aanwezi g zijn. Devrouwtje s van het pimpernelblauwtje daarentegen zetten hun eitjes onafhankelijk vand edichthei d aan

De bloemrijke bermen van de Ruidigerdreef zijn tegenwoordig het belangrijkste leefgebied voor het donker pimpernelblauwtje. De nesten van de rode steekmier Myrmica rubra bevinden zich in het hoge gras.

Theroadverges of the road Ruidigerdreef,south of the Moerputten, are atpresent the major habitat for Maculinea nausithous. Thenests of its specific host ant Myrmica rubra are restricted to the borders with high vegetation.

212 Summary/Samenvatting/Zusammenfassung

De rupsen van de pimpernelblauwtjes worden door knoopmieren meegenomen naar hun nest, ledere soort mierenblauwtje is in meer of mindere mate gespecialiseerd op een waardmierensoort.

A caterpillar of a Maculinea butterfly is taken to the nest of a Myrmica-ant. Thesurvival chance is highest in it's specific host ants' nest.

waardmiernesten af. Zij reageren opvegetati e en bloemhoofdkenmerken. Op basis vandez everschille n isd e hypothese geformuleerd,da t het donker pimpemelblauwtje welzij nwaardmie r kandetecteren ,terwij l het pimpernelblauwtje meer opvegetatie-eigenschappe n reageert. Deze hypothese is getoetst inee n experimentele opstelling in een gazenvlinderkas . Vrouwtjes van beide soorten werden vrijgelaten en konden voor de ovipositie een keuze makenui t waardplanten invakke n met Myrmica rubra, Myrmica scabrinodis ofzonde r Het vrijlaten van devlinder s in de Moerputten. mieren. Deeerst e honderd Therelease of the butterflies in the Moerputten. eiafzettingen(=oviposities)

In het oorspronkelijk leefgebied bij Krakau in Polen komen de pimpernelblauwtjes voor in een grote metapopulatie. Near Krakow in Poland the Maculinea's occur in a large metapopulation.

213 Summary/Samenvatting/Zusammenfassung

werdengeobserveer d engenoteerd . Deze latenconclusie s toeove r de reacties van devrouwtje s alszi j geconfronteerd worden met eengrat e hoeveelheid potentiele ei-afzetplaatsen. Als de beste bloemhoofdjes bezet zijn, zullen zeoo k voor de minder optimale hoofdjes kiezen.Nada t de vrouwtjes doodware nwerde n de bloemhoofdjes met kleine gazen zakjes omsloten,zoda t alle rupsen gevangen konden worden. Hierbijworde n ook de latere, minder optimale keuzes meegenomen. Uit het experiment blijkt datd evrouwtje s van beide soorten vlinders meereitje s afzetten opvlakke nwaa r hunwaardmie r voorkomt. Bij het pimpernelblauwtje worden ook de meeste rupsen opvlakke n met waardmieren gevangen. Bij hetdonke r pimpernelblauwtje daarentegen blekend e rupsenvangsten verklaard teworde n door de vegetatiestructuur en het bloeistadiumva n de bloemhoofdjes. Dit kwamdoorda t de aangeboden waardplanten inee nt evroe g ontwikkelingsstadium waren: devrouwtje s van het donker pimpernelblauwtje prefereren oudere bloemhoofdjes. Ook indi t experiment zijn dus aanwijzingen gevonden voor het belang van miereno pd eeiafzettingen , maaree n duidelijk verband werd alleen in het begin vand evliegperiod egevonden . Het pimpernelblauwtje en hetdonke r pimpernelblauwtje stellen hoge eisen aan hun leefomgeving. Het ligt ind everwachtin g datd evlinder s de plekkendi e aan hunecologisch e eisenvoldoen ,kunne n selecteren.I n tegenstelling tot desituati e ind evlinderkoo i isi n hetvel dd e waardplant Sanguisorba officinalisi n hogedichthei d aanwezig,terwij l de waardmieren minder algemeen zijn. Dit houdt dus inda t een deelva n de waardplanten ongeschikt isvoo r de ei-afzet (=ee n "sink"vertegenwoordigen) , omdatd e mieren een beperktfoerageergebie d hebben endez e planten niet bereiken. De rupsenworde n niet door mierengevonde n engaa n dus dood.Voo r devlinder s zijn alleen die planten echte waardplanten ("resource") die binnen hetfoerageergebie d van een mierenneststaan . Alleen de rupsen opdez e planten hebben een kanso mdoo ree nwerkste r gevonden engeadopteer d teworden . Heteffec t vand e aan-o f afwezigheid van mierennesteno pd eeiafzettin g van devrouwtje s en het voorkomenva n de populaties werd bestudeerd op meerda n 600 proefvlakken ind e Moerputtene nd eomgevin g ervan.O pdez e plots werden vegetatiesamenstelling,vegetatiestructuur , mierenfaunae n microklimaat indetai l gemeten enbeschreven .Ovipositie s van hetdonke r pimpernelblauwtje zijngemakkelij k aant etone n door deeirestante n opd e bloemhoofdjes van dewaardplan t tetellen ,nada t deze eind September zijngeplukt . Deze methodevoldee d nietvoo r hetpimpernelblauwtje , zodat bij deze vlinder vrouwtjes gedurende de eerste helftva n devliegtij d

214 Summary/Samenvatting/Zusammenfassung

werden gevolgd.Bi j beide soorten prefereerden de vrouwtjes waardplanten ind e buurtva n Myrmica-nesten voor deovipositie . Vervolgens werdd e habitatselectie op een hoger ruimtelijk schaalniveau getoetst door deverspreidin g van devlinder s sinds 1991pe r plott e analyseren.Oo k opdi t schaalniveau werd selectie voor plots met mierenaangetoond .Allee n injaren ,waari n de populatiedichtheid vand e vlinders hoogwas ,werde n ook plots met geschikte vegetatie maarzonde r mieren bezet. Op basisva n deze resultaten kunnen we ervan uitgaan,da t de eiafzetting van pimpernelblauwtje endonke r pimpernelblauwtje afhankelijk van mierenaanwezigheid plaats vindt. Eitjes worden bij voorkeur dicht bij mierennestenafgezet , en hetsucce s vand e herintroductie iszeke r ten delet e danken aan ditgedrag .

Het isopvallen d datondank s deselecti e van proefvlakken met beide voedselbronnen voor de rupsen,d e kolonisatie vangeschikt e plekkeno p hogere ruimtelijke schaal beperkt blijft. Geschikte plekken opgrat e afstand van bezette plekken blijken minder gekoloniseerd teworde n dan verwacht. Daarentegen worden plots metee n slechte habitatkwaliteit op korte afstand van bezette plots gemakkelijk bezet. Beperkingen ind e verbreidingscapaciteit op individueel niveau belemmerend e habitatselectie. Dit heeft een effect op de kolonisatie en hetverlate n van proefvlakken van degehel e populatie, endu soo k in het aangetroffen patroonva nvoorkomen . Het donker pimpernelblauwtje koloniseert over grotere afstanden dan het pimpernelblauwtje, maar beide soorten zijntoc h zeer honkvast. Bijhe t pimpernelblauwtje latend e kolonisaties en lokale extincties een patroon van uitbreiden enterugtrekke n binnend e heterogene plotso p de Bijenwei zien, hetenig e bezette hooiland.Voo r de populatie als geheel zijn er consequenties verbonden aandez e beperkte verbreiding. De hoge habitatkwaliteit voor het pimpernelblauwtje enzij n waardmier Myrmica scabrinodis opd e Bijenwei moet behouden blijven. Dezevlinde r kan hooguit naburige hooilanden bereiken,terwij l datove r grotere afstanden niett everwachte n valt. Het donker pimpernelblauwtje daarentegen kanwe l een metapopulatieopbouwe n ind e omgeving vand e Moerputten. Dit houdt inda t niet alleen in het natuurgebied maar ook op wegbermene nslootrande n ind eomgevin g bij inrichtings-e n beheersmaatregelen met de habitateisen van deze vlinder, zijn waardplant enwaardmie r rekening gehouden moetworden .

Vijfjaa r nad e herintroductie werdee n genetische analyse vand e bronpopulatie in Polene nd egeherintroduceerd e populatie ind e

215 Summary/Samenvatting/Zusammenfassung

Het donker pimpemelblauwtje heeft een voorkeur voor een ruigere vegetatie dan het pimpemelblauwtje, zoals hier op de noordzijde van de spoordijk in de Moerputten. M. nausithous prefers a tall herb vegetation, such as on the railway embankment in the Moeputten.

Moerputten uitgevoerd. Bijbeid e geherintroduceerde populaties zijnd e aantallen vlinders nad evrijlatin g niet sterkteruggevallen .D e populatiegroottes zijnvoldoend e snel toegenomen ene rwer dallee n een gering verlies aangenetisch e variatie vastgesteld. De bronpopulatie had slechtsweini g genetische variatie (detecteerbaar door eiwit- electrophorese), maare rwer d geen negatief effect hiervan op devestigin g en uitbreiding vand e geherintroduceerde populaties vastgesteld. Blijkbaar warend e aantallen vrijgelaten vlinders end e kwaliteit van de herintroductie-locatie vangrote r belang voor het herintroductiesucces dand egenetisch e variatie vand e bronpopulatie.

Hetjuist e beheer van de bermen bepaalt voor een belangrijk deel de overlevingskansen van het donker pimpemelblauwtje. Als er op het verkeerde moment gemaaidword t zijn er geen waardplanten beschikbaar als de vlinders vliegen. Bij aangepast maaibeheer kan moderne landbouw samen gaan met kwetsbare vlindersoorten.

Theright management of the roadverges is an important factor in the survival of Maculinea nausithous. Ifsites are mown at the wrongmoment no larval foodplants are available for oviposition. But with small adaptations in the management, modern agriculture and vulnerable butterfly species can occur side by side.

216 Summary/Samenvatting/Zusammenfassung

De inonbrui k geraakte spoorbrug over het meertje in de Moerputten.

Therailway bridge over the centrallake in the Moerputten nature reserve is not in use anymore.

Bij beide Maculinea soortenwerde n alleen voor een bepaald eitwit (Aco-K) genetische verschillen tussen bran-e n herintroductiepopulatie gevonden.I n combinatie met deverplaatsin g vand e vliegperiode end e korte levensduur vand e vlinders leidt ditto td e conclusie datd e

Rondom het meer komt nu veel wilgen- en elzenbroekbos voor, waar vroeger blauwgraslanden waren, vergelijk fig. 1.10, pag. 37.

At present moist forests occur on the former meadows around the lake, compare fig. 1.10, page 37.

Niet alle ogenschijnlijk geschikte hooilanden met veel grote pimpernel worden gekoloniseerd. Dat kan worden veroorzaakt door de afwezigheid van Myrmica- mieren, of door een te grote afstandto t een bezette plek.

Not all meadows with lots of Sanguisorba officinalis are colonised. Thehost ant species might be absent or the distance to an occupiedplot is toolarge to bridge with normal dispersal behaviour.

217 Summary/Samenvatting/Zusammenfassung

geherintroduceerde populaties opd e nieuwelocati e selectie hebben ondervonden. Deze conclusie wordt ondersteunt door deanalys e vand e vleugelpatronen van bronpopulatie, Moerputten-populatie, de historische Nederlandse populaties en populaties in Frankrijk, Belgie en Duitsland.D e groep bestaande uitd e oude Nederlandse, Belgische en Franse populaties verschilt inzij n uiterlijk vand egroe p bestaande uitd e Poolse, Duitse end e Moerputten populaties. Gesteld kanworde nda tdi t het gevolg isva n verschillende ijstijdrefugia tenweste n ente nooste n vand e Alpen. Bijd e herintroductie zijnvlinder s van een bepaalde andere biogeografische oorsprong verplaatst naar een plek buiten huno p natuurlijke wijze gekoloniseerde verspreidingsgebied. Deverplaatst e vlindersware naangepas t aand e klimatologischeomstandighede ni nhe t zuiden van Polene n hebben selectiedruk ondergaan op hun nieuwe vliegplaats. Beide geherintroduceerde populaties hebben een negatieve inteeltcoefficient F,s.Di twi lzegge n datzi j opee n of ander manier instaa t zijn omee n hogere mate vanvariati e te handhaven dano p basis van toevallige paringen verwacht kanworden . Het is nietduidelij k hoedi t phenomeentotstandkomt , enverde r onderzoek isnodi go mdi t opt e helderen.

Voor de levensvatbaarheid op langetermij nzoude nd e geherintroduceerde Maculineapopulatie s metapopulaties moetenkunne n ontwikkelen,zoda tz e beter beschermdzij ntege ndemografische , genetische enomgevingscatastrofes . Ook alzij nd evlinder s instaa to m plekken met hunwaardmiere no pt e sporen,hu nverbreidin g en kolonisatievermogenzij nzee r beperkt. Het pimpemelblauwtje isallee n in staat omee n metapopulatie binnen degrenze n van het natuurreservaat opt e bouwen. Hiervoor moette n eerste de hoge habitatkwaliteit op het bezette hooiland door constant beheer bewaard worden. Daarnaast moeten naburige hooilanden opee nvergelijkbar e wijze beheerd worden zodat zijgekoloniseer d kunnen worden. Uitbreiding naar andere natuurgebieden isallee n door middelva n herintroducties mogelijk. Het donker pimpemelblauwtje isvoo r hetopbouwe n van een populatienetwerk afhankelijk vand ewegberme ne nslootrande n ind e omgevingva n het natuurreservaat. Dezesoor t isdu szee rgevoeli g voor hetbehee rzoal s dat door lokale overheden enaanwoner s wordt uitgevoerd. In het kader van het Convenant Pimpernelblauwtjes zijn beheersveranderingen uitgevoerd en isaangetoond ,da t de mierenfauna opdez ewijz e verbeterd kanworden . Maartegelijkertij d isoo k duidelijk geworden,da t het

218 Summary/Samenvatting/Zusammenfassung

leefgebied vand eMaculinea's doo r illegaleactiviteite n gemakkelijk vernield kanworden .Oo k het donker pimpernelblauwtje kangebiede n op grotere afstand nietzelfstandi g bereiken enzo udaa r geherintroducerd moetenworden .

Samenvattend:Vee lleefgebiede n vand e pimpernelblauwtjes zijn doortoedoe n vand e mensverdwenen . Resterende terreinenzij n achteruitgegaan inkwaliteit . Hierdoor endoo r beperkingen inhe t verbreidingsvermogenva n beidevlindersoorte n worden populaties op langetermij n in hun levenvatbaarheid aangetast. Omdatd e meeste natuurreservaten maar kleinzij n en ingebed zijn inagrarisc h landschap, moeten devlinder s de reservatenwe lverlate n enzic h ertussen vestigen enverbreiden . Hunoverlevingskan s isdaa r sterk gebonden aanhe t landgebruik. Als herintroductieprojektenvoorzichti g eno p wetenschappelijke basisworde n uitgevoerd, kunnen zij belangrijk zijnvoo r de natuurbescherming. Negatieve effecten opgrot e schaal,zoal ste n gevolge van klimaatsverandering, kunnenerme ete ndel e worden gecompenseerd. Maar hoezorgvuldi g dewetenschappelijk e uitvoering ook is,herintroductieprojekte n hebben een lagekan sva n slagen als niet ook gedegen voorlichtingscampagnes naar de lokale bevolking,d e lokale autoriteiten, natuurbeschermingsorganisaties en natuurbeheerders worden meegenomen.

219 Summary/Samenvatting/Zusammenfassung

Zusammenfassung

InEurop a kommenfun f Blaulingsartende r Gattung Maculinea vor. Durch ihre besondere Okologieal s Parasiten inde n Nestern von Myrmica- Ameisenerfreue nsi esic hde r besonderenAufmerksamkei tviele r Lepido- pterologen.All efun fArte nzeige n innerhalb ihres Verbreitungsgebietes einen starken Ruckgang an Populationen wie auch vielfach bezuglich ihrer Populationsgrossen. Diese Ruckgangewerde n hauptsachlich durch landwirtschaftliche Nutzungsintensivierung einerseits sowieda s Brachfallen von Extensiv-Grunland andererseits verursacht. Inbeide n Fallenander n sichdi e Lebensraume der Falter grundlegend.Zude m werden Lebensraume durch Siedlungsbau, Infrastrukturmassnahmen, Aufforstungen und Entwasserung zerstort. Die verbleibenden Populationen werden durch Fragmentierungihre r Lebensraume und Populationensisolation beeintrachtigt.

Der HelleWiesenknopf-Ameisenblaulin g (Maculinea teleius)un dde r DunkleWiesenknopf-Ameisenblaulin g (Maculinea nausithous)besiedel n Feuchtwiesen,feucht e bisfrisch e Mahwiesen undjunger e Brachen. Wiesenmi tVorkomme nde s Hellen Wiesenknopf-Ameisenblaulings miissen,j e nach Produktivitat, ein-bi szweima ljahrlic h gemahtwerden . Der DunkleWiesenknopf-Ameisenblaulin g toleriert daneben auch Brachen,di e nurall edri e bisfiin f Jahregemah twerden . Inde n Niederlanden kamen beideArte n auf Wiesen entlang von Flussen und Bachen inde n Provinzen Limburgun d Nord-Brabant vor. Inde n siebziger Jahren verschwanden die letzten Populationen.

ImRahme neine sWiedereinburgerungs-projekte s wurden imJahr e 1990 Falter beiderArte n im Naturschutzgebiet Moerputten inNord - Brabantfreigelassen . DieWiedereinburgerun g wurde nachde n Regeln der IUCN (International Unionfo r Conservation of Nature and Natural Resources) ausgefuhrt. Esstellt e sich heraus,das s dieses Naturschutzgebiet der einzig moglicheOr t inde n Niederlanden fur ein derartiges Projekt war, da nur hier dieWirtspflanz e Sanguisorba officinalis in hoher Dichtevorkomm t und beideWirtsameise n (Myrmicarubra un d Myrmica scabrinodis) nachgewiesen werden konnten.Di e freigelassenen Falter stammenau s grossen Metapopulationen imTa lde s FlussesWisl a inde r Nahe des Dorfes Kostrza sudlich von Krakau in Polen. Die Metapopulationen bestehen aus mehrerenTausen d Faltern proJah r und wurden durch dasWegfange n der Falterfu r das

220 Summary/Samenvatting/Zusammenfassung

Wiedereinburgerungsprojekt nicht beeintrachtigt.

Nachde rWiedereinburgerun g zeigten beideArte n unterschiedliche Populationsentwicklungen. Der HelleWiesenknopf-Ameisenblaulin g nahm inde nfolgende n drei Jahren zu.Anschliessen d nahmdi e Population stark ab,wa s moglicherweisedurc h Uberbelastungde rAmeisenneste r verursachtwurde ,u msic hdanac h auf eineAnzah l von rond30 0 Faltern proJah r einzupendeln. Dieser Prozess wiederholtesic h noch einmal,d a 1995durc h einen unglucklichen Zufall das Habitat imAugus t gemaht wurde. DieAr t hat sich bislang raumlich nicht ausgebreitet. Bis heute kommtsi e nur auf der einenWies e vor, auf derdi e Falter ursprunglich freigelassenwurden .

Der Dunkle Wiesenknopf-Ameisenblauling durchlebte imJah r nachde r Wiedereinburgerung zunachst einenstarke n Ruckgang, bevor die Anzahl der Falter inde nfolgende n Jahren stieg. DieSchmetterling e habendi e Wiese,au f der siefreigelasse n wurden,verlasse n und sich aufde m Bahndamm,de r mittendurc h das Naturschutzgebiet lauft,angesiedelt . Es stellte sich heraus,das s hier die hochste Dichtede r spezifischen Wirtsameise Myrmica rubravorkam . Nachzwe i Jahrenwurde n die Boschungen der Strasse Ruidigerdreef ande r Sudseite des Naturchutzgebietes kolonisiert. Diese zweite Subpopulation breitete sich relativ schnell inAnzah l und Flache aus. Beide Subpopulationen liegen mehral s 500 Meter von einander entfernt. ImRahme nvo nFang - Wiederfangstudien zeigte sich, dasszwische n 8un d 10%de r wiedergefangenen Falter inde rjeweil s anderen Subpopulation markiert wurden.Wiede r zweiJahr e spater etablierte sich einedritt e Subpopulation ineine mAbstan d von mehr als 5km ,di ejedoc h immer noch sehr klein ist.

Trotz der imallgemeine n positiven Resultate zeigten sich einige besorgniserregende Fakten.Beid eArten ,besonder s der Helle Wiesenknopf-Ameisenblauling, haben eine kurze Lebenserwartung, wodurch sie empfindlichgegenube r stochastische Katastrophen sind. Ausserdem hat sichdi e Flugzeit derwiedereingeburgerte n Populationen, verglichen mitde n Mutterpopulationen,z u einemfruhere n Zeitpunkt hin verschoben. Dieszeigt e sich auch imVergleic h mit den Flugzeiten der historischen hollandischen Populationen.Aktuel lflieg t der Helle Wiesenknopf-Ameisenblauling vonAnfan g bis EndeJuli , der Dunkle Wiesenknopf-Ameisenblauling von Mitte Juli bis MitteAugust . Beide n fruheren hollandischen Populationen und in Kostrza dauert die Flugzeit

221 Summary/Samenvatting/Zusammenfassung

beiderArte n bis EndeAugus t undlanger .

Die Larven der Wiesenknopf-Ameisenblaulinge nutzenzwe i verschiedene Ressourcen,di eWirtspflanz e Sanguisorba officinalisun d eine,j e nachAr t unterschiedliche, Myrm/'ca-Wirtsameisen. Die Eier werdeneinzel n oder zu mehrerenau f die Blutenkopfchen des Grossen Wiesenknopfes abgelegt, wobei die Falterarten verschiedene phanologische Stadiende r Blutenkopfe bevorzugen. Die L1- bisL3 - Raupenernahre n sich von derWirstpflanze , auf der siezwe i bisdre i Wochenverbleiben . Dann lassen sie sichz u Bodenfallen ,w osi e vonde n Arbeiterinnen unterschiedlicher Myrm/ca-Artenadoptier t und indi e Ameisennester mitgenommen werden. ImAmeisennes ternahre n sichdi e Raupen parasitisch vonAmeisenlarven . Hierfindetdie Oberwinterung und imfolgende n Fruhjahr dieVerpuppun g statt. Der DunkleWiesenknopf - Ameisenblauling parasitiert inde n Nestern von Myrmica rubra,wahren d der HelleWiesenknopf-Ameisenblaulin g hauptsachlich inde n Nesternvo n Myrmica scabrinodis zufinde n ist. DieArbeiterinne n derAmeisenvolke r bleiben imallgemeine n immer inde r Nahe des Nestes undentferne nsic h selten mehral s2 Meter von diesem. Da inde n meisten Blaulings- Lebensraumende r Grosse Wiesenknopf in hoher Dichtevorkommt , wahrend dieAmeisenneste r vielseltene r sind, ist nur ein Teilde r Wiesenknopfpflanzen alsWirtspflanze n geeignet, der im Fouragierbereich einesAmeisenneste s liegt. DieWeibche n der Blaulinge hattendaru m einengrosse nVorteil ,wen n sie ihre Eier nurau fsolche n Pflanzen ablegen wurden,di e aufgrund derAnwesenhei t der spezifischenWirtsameis e ihre Funktion alsWirtspflanz e erfullen konnen.Allerding s wird bisher davon ausgegangen,das sdi e Macu//nea-Weibchen ihre Eier in Bezugau f die Ameisenwillkurlic h ablegen (=random oviposition hypothesis).De r phanologische Zustand des gewahlten Blutenkopfchens unddi e Eigenschaften der Vegetation inde r direkten Umgebungseie nfu r das Weibchen von ausschlaggebender Bedeutung.Wi r haben getestet, obda s tatsachlich der Fall ist,ode r obda sWeibche n nebende n Eigenschaften des Blutenkopfchens und derVegetatio n auch auf dieAnwesenhei t bzw. Dichte vonAmeisennester n reagiert (=ant-mediated oviposition).Hierz u wurde die Eiablage-Dichte auf Probeflachen mit unterschiedlichen Wirtspflanzen- undWirtsameisendichte n inde r Umgebung des Dorfes Kostrza verglichen. Eszeigt e sich, dass die Weibchen des Dunklen Wiesenknopf-Ameisenblaulings sich bevorzugt dort aufhalten und Eier ablegen,w o die Nestdichte der Wirtsameise Myrmicarubra hoc h ist. Bei dem HellenWiesenknopf-Ameisenblaulin g konntejedoc h keine Verbinding

222 Summary/Samenvatting/Zusammenfassung

zwischen Eiablageorten undWirtsameisendicht e gefunden werden.Hie r waren die Eigenschaften der Vegetation undde r Blutenkopfchen wichtiger. Aufgrund dieser Unterschiede stellenwi r die Hypothese auf, dass der Dunkle Wiesenknopf-Ameisenblauling dieAnwesenhei t seiner Wirtsameisen feststellen und darauf reagieren kann,wahren d der Helle Wiesenknopf-Ameisenblauling beide rAblag e der Eierau f Vegetationseigenschaften reagiert.

Diese Hypothese wurde ineine m Insektarium experimentellgetestet . Weibchen beider Falterarten konnten beide r Eiablagefre i zwischen Wirtspflanzen mit Myrmica rubra,Myrmica scabrinodis oderohn eAmeise n invergleichbare r Vegetation wahlen. Erstwurde n die Eiablagen bobachtet und notiert. Spater wurden die Blutenkopfchen in kleine Sackchen eingeschlossen, urndi e Raupenz ufangen . Die Eiablage-Beobachtungen erlauben Ruckschlusse auf dasVerhalte n derWeibchen ,wen n ihnen ein Ubermassa n unbelegten Wirtspflanzen zur Verfugung steht. Beide n Raupenfangen werden auchdi e Eiablage-Entscheidungen einbezogen, als bereits einTei lde rWirtspflanze n belegt war. Bei beiden Maculinea- Arten spielte zuAnfan g der Fluzeit dieAnwesenhei t derjeweilige n Wirtsameise einegross e Rollefu r die Eiablagen. Beide m Hellen Wiesenknopf-Ameisenblauling zeigten auchdi e Raupenfange, dass die Weibchen vorzugsweise Wirtspflanzen in Probeflachen mit Ameisen belegten. Beide m DunklenWiesenknopf-Ameisenblaulin g liesssic h auch ein Einfluss derVegetationseigenschafte n nachweisen,de r darauf zuruckzufuhren war, dass dieangebotene n Wirtspflanzen zujun gwaren . Folglich gibt eserst e Hinweise,das sfii r beideAlle n dieAnwesenhei t der Wirtsameisen beide rWah leine s Blutenkopfchens furdi e Eiablage eine Rolle spielt. Dieser Effekt istz u Beginn der Flugzeit deutlich ausgepragt, wahrend spater dieVegetationsstruktu r undde r phanologische Zustand des Blutenkopfchens gleichfalls von Bedeutung sind.

Wie bereits zuvor ausgefuhrt wurde, konnen dieAmeisenblauling e aufgrund ihrer spezifischen Okologie nur ein Teil der ihnen potentiell zur Verfugung stehenden Wiesenknopfpflanzen sinnvoll nutzen. Pflanzen ausserhalb derAktionsradie n vonAmeisennester n stellen Populationssenken dar, dadi e hier befindlichen Raupen keine Oberlebenschance haben. Die Bedeutung derAn -un dAbwesenhei t von Ameisennestern auf die Eiablage und dieAnwesenhei t von Faltern wurde im Freiland anhand von mehral s60 0 Probeflachen im Naturschutzgebiet Moerputten undseine r Umgebung untersucht. Eiablagen des Dunklen

223 Summary/Samenvatting/Zusammenfassung

Wiesenknopf-Ameisenblaulingswurde n anhandvo n Eischalenresten auf den Blutenkopfchen des GrossenWiesenknopfe s nachgewiesen.Di e Blutenkopfchen wurden Ende September gesammelt. Dadies e Methode fur den HellenWiesenknopf-Ameisenblaulin g nicht geeignet war, wurden dieWeibche n dieser Art inde r ersten Halfte der Flugzeit direkt beide r Eiablage beobachtet. DieWeibche n beiderArte n legten ihre Eier vorzugsweise auf Blutenkopfchen inde r Nahevo n Wirtsameisennestern ablegen. Die Habitatwahl der Falterpopulation wurde untersucht, indem dieAn -un dAbwesenhei t der Falter aufde n Probeflachen seit 1991 analysiert wurde. DieZusammensetzun gde rVegetation ,di e Vegetationsstruktur, dieAmeisenfaun a und das Microklima wurden erfasst bzw. gemessen. Die Resultate zeigten,das s sichauc h dieadulte n Falter bevorzugt auf Flachen mitAmeise naufhielten . Nur inJahre n mitseh r hoher Falterdichte wurden auch Flachen mit geeigneten Vegetationseigenschaften, wo aber dieAmeise n fehlen,besetzt . Diese Resultate bestatigen,das s die Falter ihreWirtsameise nentdecke n konnen und die Eiablage nicht zufallig stattfindet. Der Erfolg des Wiedereinburgerungsprojekt.es ist unter anderemdarau f zuruckzuftihren, dass Verluste durch zufallige Eiablage vermiedenwerden .

Esbleib terstaunlich ,das sdi e Falter beiderArte noffenba r inde r Lage sind, Flachen mit beiden larvalen Futterquellen aufzuspuren, aber dennoch auf Landschaftsebene Flachen hoher Habitatqualitat unbesetzt bleiben. EineAnalys e derAbstand e zwischen besetzten und unbesetzten Patcheszeigte ,das s Patches mit hoher Habitatqualitat unbesetzt bleiben, wennde rAbstan d zum nachsten besetzten Patchz u gross ist. Patchvo n niedriger Habitatqualitat werden hingegen schnell kolonisiert,wen n sie nahe bei besetzten Patches hoher Habitatqualitat liegen.Limitiert e Dispersionskapazitaten fuhren daher zu Einschrankungen inde r Habitatwahl der Population. Der Dunkle Wiesenknopf-Ameisenblauling kannzwa r grossere Strecken zurucklegen als der HelleWiesenknopf - Ameisenblauling,doc h bei beidenArte n werden geeignete Lebensraume ingrosse mAbstan dz u besiedelten Flachen nicht kolonisiert. Beim Hellen Wiesenknopf-Ameisenblauling beschreibt das Kolonisations-und Extinktionsmuster einenAusbreitungs - und Ruckzugsprozess innerhalb der einzigen besetzten, heterogenen Mahwiese BW. Die eingeschrankte Dispersionsfahigkeit fuhrtz u Konsequenzen fur die Populationen. Furde n HellenWiesenknopf-Ameisenblaulin g und seineWirtsameis e Myrmica scabrinodis ist es dringend erforderlich,das s die hohe Habitatqualitat auf der Mahwiese BWerhalte n bleibt. DieseAr t kann hochstens benachbarte

224 Summary/Samenvatting/Zusammenfassung

Mahwiesen erreichen,wahren d langeWanderstrecke n nur selten zuruckgelegt werden. Fur den DunklenWiesenknopf-Ameisenblaulin g sollten dieVoraussetzunge n geschaffen werden,ein e Metapopulation aufbauen zu konnen.Hierau s folgt, dass das Biotopmanagement nicht nur innerhalb des Naturschutzgebiets, sondern auch ausserhalb an Strassenboschungen und Bachufern geandert werdensollte .

FunfJahr e nachde rWiedereinburgerun g wurde ein genetischer Vergleich der Mutter- undTochterpopulatione n mit Hilfevo nAllozym - Electrophorese durchgefuhrt. Beide nwiedereingeburgerte n Populationen beider Arten konnte "Flaschenhals" infolge der geringenAnzah la n GrCinderindividuen nachgewiesen werden. Die Individuenzahl hati n beiden Populationen schnell zugenommen, und es konnte nurei n geringfiigiger Verlust angenetische r Variation entdecktwerden . Bereits die Mutterpopulationen wiesen einegering e genetische Variabilitat auf, was allerdings keinen negativen Einfluss aufde n Wiedereinbiirgerungserfolg hatte.Offensichtlic h habendi eAnzah lde r Grunderindividuen unddi e Habitatqualitat des neuz u kolonisierenden Gebietes einen grosseren Einfluss auf den Erfolg alsdi e genetische Konstitution der Mutterpopulation.

Nachde rWiedereinburgerun g hat bei beidenArte n nur auf dem Locus Aco-K einegenetisch e Differenzierung gegenuber der Mutterpopulation stattgefunden. Dasic hdi e Flugzeit verschoben unddi e Lebenserwartung der Falter abgenommen hat, kommenwi r zu der Schlussfolgerung, dass die Grunderpopulation unter erheblichemSelektionsdruc k durchdi e veranderten Umweltbedingungengestande n haben muss. Diese Schlussfolgerung wird unterstutzt durchdi eAnalys e der Fleckenmuster auf den Flugelunterseiten. Indies e Studie wurden ausser Mutter- und Tochterpopulation auch die historischen hollandischen Populationen und Populationen aus Frankreich, Belgien und Deutschland einbezogen.I m Phanotyp unterschied sich die Gruppe aus den historischen hollandischen, den belgischen undfranzbsische n Populationen von der Gruppe der polnischen,deutsche n und der Moerputten-Populationen. Diese Unterschiede sindwahrscheinlic h auf unterschiedliche eiszeitliche Refugien westlich undostlic h derAlpe nzuruckzufuhren . Offensichtlich wurden Tiere eines bestimmten biogeographischen Ursprungs inda s Arealde r Populationen eines anderen biogeographischen Ursprungs transportiert. Die Grunderpopulation,di ea n die klimatologischen Verhaltnisse in Polenangepas t war, wurde dadurch veranderten

225 Summary/Samenvatting/Zusammenfassung

Selektionsbedingungen ausgesetzt. Bei beiden Grunderpopulationen wurde ein negativer Inzuchtkoeffizient festgestellt. Also sind die Populationen inde r Lage,ein e hohere Heterozygositat zu bewaren, alsaufgrun d von Zufallspaarungen erwartet werden kann.E s ist unklar,mi twelche m Mechanismusdiese s Phanomen erklart werden kann,weiter e Studien mussen dieszeigen .

Zur Erhohung der langerfristigen Uberlebenswahrscheinlichkeit empfiehlt es sich, denwiedereingeburgerte n Macu//nea-Populationen Moglichkeiten zur Entwicklung von Metapopulationenz u bieten,s odas s sie mogliche negative Auswirkungen von demographischen und genetischen Zufallseffekten sowie der Umweltstochastizitat kompensieren konnen. Die Falter sind zwar inde r Lage,geeignet e Lebensraume (inklusive Wirtsameisen)z u entdecken,doc h ihre Dispersionsfahigkeit ist begrenzt. Der HelleWiesenknopf-Ameisenblaulin g kann innerhalb der Grenzen des Naturschutzgebietes eine Metapopulation aufbauen.Hierz u musseinerseit s die hohe Habitatqualitat auf dereinzige n besetzten Mahwiese durch konstantes Biotopmanagementerhalte n bleiben, andrerseits mussen benachbarte Mahwiesen ingleiche r Weise bewirtschaftet werden,s odas s auchdies e erfolgreich kolonisiert werden konnen.Ander e Naturschutzgebiete werden mitgrosse r Wahrscheinlichkeit nicht besetzt werden,e s sei denn durch weitere Wiedereinburgerungsprojekte. Der Dunkle Wiesenknopf-Ameisenblauling besitzt die Dispersionskapazitat, selbststandig eine Metapopulation mit Habitatverbund aufzubauen. Hierbei iste r abhSngigvo n der Habitat­ qualitat der Strassenboschungen und Bachrander inde r Umgebungde r Naturschutzgebiete, dadies e alsTrittsteinbiotop e genutztwerden . Daher kanndies e Art leicht durch das Management dieser Landschaftselemente geholfen werden,wi e esdurc h offentliche Organisationen undAnraine r ausgefuhrt wird. Ein lokalbegrenzte s Macu//nea-Hilfsprogrammha t gezeigt, dass im Rahmeneine s offentlich ausgefuhrten Biotopmanagements dieAmeisenfaun a auf Boschungen nachde n dkologischenAnforderunge n der Blaulinge entsprechend verbessert werden kann.Leide r wurde aber gleichzeitig auchdeutlich ,das s Waciy//nea-Habitatleich t durch illegale Aktivitaten zerstortwerde nkann . Auchfu r den Dunklen Wiesenknopf-Ameisenblauling gilt,das s die Kolonisation neuer Lebensraume uber grossere Abstande nur durch Wiedereinburgerungsprojekte erfolgen kann.

Wir konnen zusammenfassen, dass durch die limitierte Dispersions-

226 Summary/Samenvatting/Zusammenfassung

kapazitat undanthropoge n verursachte Habitatverschlechterungenun d- verluste die langerfristige Uberlebenswahrscheinlichkeit der Maculinea- Populationen vermindert wird. Dadi e meisten Naturschutzgebiete klein sind undal s isolierte Inseln inde r agrarisch gepragten Landschaft liegen, miissen die Falter sie auf Dauer verlassen undsic h dazwischen ansiedeln undverbreiten . Ihre Uberlebenswahrscheinlichkeit istdan n ingrosse m Masse abhangig von der Landnutzung.Wen n Wiedereinburgerungsprojekte umsichtig undau fwissenschaftliche r Basis ausgefuhrt werden, konnen sie einewichtig e Rolleal s Instrument des Naturschutzes spielen. Negative Effekte grossflachiger Umweltveranderungen,wi ez.B . Klimaerwarmung, konenau fdies eWeis e unter Umstandengemilder t werden.Abe rwi esorgfalti g die wissenschaftliche Ausfuhrung auch ist,Wiederburgerungsprojekt e sind erst dann langfristig erfolgreich,wen n Informationscampagnenfu r die lokale Bevolkerung,offentlich e Organisationen, Naturschutzorganisationen und Naturschiitzer inda s Gesamtprojekt einbezogenwerden .

227 Summary/Samenvatting/Zusammenfassung

228 Thankyou, dankje wel,Danke, gracias!

Dit boekje draagt slechts mijn naam, maard ebasi s ervan konallee n stevig zijndoo r de bijdragen vanvelen : mijnfamilie ,vrienden , studenten encollega's . Erzijn honderden vlinders gevangen en eitjes geteld, duizenden bloemhoofdjesgemeten , honderdenvegetatie-opname n gemaakt en mierengevangen ,e ne r is nogzovee l meergebeur d door en dankzijjullie ! Thank you, dankje wel, Danke,gracias ! Als eerstewi l ik diegenen noemen,di e mij het meest hebben moeten missene nwien s bijdrage ook daardoor hetgroots t isgeweest : Chris, Eva en Bosse. Bossefang tjede n Schmetterling, die schnellen ofta m leichtesten. Evafinde t Pflanzen interessanter, sie entdeckt auch die kleinsten vegetativen Enziane.Chri swa s er gewoon altijd. Erzij n geen woorden,o m mijnwaarderin g voor mijn kleingezinnetj e op passende wijze te omschrijven.Vana f nuza l er meertij dvoo r ons alien samenzijn . Mutti und Paps,viele n Dankfu r Eure Unterstutzung und Euer Interesse. Dies ist auch Euer Buch,auc h Euer Erfolg.Und ,Mutti ,ohn e Dichwar e es noch immer nichtfertig . Herbert Prins,d eeerst e keer hebj e meverbaasd ,toe nj e mijn promotorwild ezijn , endaarn a volgden nogvel e momentenva nverbazing . Jij maakteee n belangrijk deel uitva nd ewetenschappelijk e omgeving, die ik nodig had,o m mijn Maciv/;nea-projectt e kunnen voortzetten. Daarnaast wistj e ook een gezelligewerksfee r enee n vertrouwde omgeving te creeeren,waari n ik me konontplooien . Het bespreken vonopera' s opee n gammele brug meters boven het meertje ind e Moerputten was een bijzonder genoegen. Maar nog leuker waren dediscussie s over vraagstellingen ind eecologi e enove r selectie. Grote buffels en kleine vlinders mogenwe l erg op elkaar lijken,toc hzij n er belangrijke verschillen. Bedankt voor de mooietij d bijd egrot e grazende viervoeters! Paul Brakefield,than k you very much,oo kji j wilde voor med e rolva n promotor opj e nemen.Jou w steunwa svoora l in het begin enhe t einde belangrijk, en daartussen kon ik altijd bijj e terecht om met named e vraagstukken ind egenetic a opt e lossen. Ik kon inj e labwerke n enw e zaten samen achter decompute r om het kleine beetje orde ind e vleugelpatronen teontdekken .Verde r was er dewonderbaarlijk e reis,di e devlindervleugeltje s maakten. Enbovendie n zijnw e allebei vlindergek .... Janva n der Made,zonde rjo u zou ergee n herintroductie zijn geweest, en later geen promotie.Toe nj e mij indiens t nam,wa s alles al opgestart. Samen reisden we naar Poleno mvlinder s te halene n in Nederland vrijt e laten. Jij had eindeloos veelvertrouwe n erin dat hetallemaa l goedzo u gaan. Later gafji j meee nvast e baan van een dag perweek , zodat ikoo k opd evakgroe p konwerken . Dankje wel,Jan ! Tim Pavlicek, mijnvlindermaatj e vanaf het begin.Ji j hielp mij met ontzettend veel kleine dingen,di e ieder voor zich onbelangrijk lijken,maa r

229 alle bijelkaa r zeer belangrijk zijn. Hetmees t belangrijke was,da tji j er gewoonwas , ind eoud e maar meer nog ind e nieuwe vakgroep. Ik benz o blij, datj e er nual s paranimfwee r bent! Dewild etropenjongen s en-meisje sva n de Bornse steegzij n eener g gezellige, creative eneigenwijz e club. Met Ignas, Pieter,Arend ,Sip , Christien, Max, Lalith,Johan ,Ha ne nTjakki e neb ik menigevraa g besproken. Desamenwerkin g met Frank gedurende het laatstejaa r was bijzonder prettig enleerzaam . Paul (bedankt voor dedatum!) , Margje, Claudius, Bas, Pirn, Liesbeth, Michael,Barend ,Karin ,Patric k en Fulco deelden mijn promotie perikelen.A t this place Iwoul d liket oad d a special thank yout o mydancer s of the Deadline Blues:Shem , Charud and Theodore. Iha d manyjoyfu l moments while dancing this blues together with you.M yfrequentl y changing room mates also deserve many thanks for inspiring discussion andfo r helping me remember mystil l poor Spanish. Herman enAld o hielpen me,d e problemeno pt e lossen met mijn computer, die ikweini g liefdevol "het kleitablet" noemde. Gerda zorgde voor mijn budget enWillemie n ondersteunde mijvanui t het secretariaat. Dankj e wel allemaal!Oo k deoverig e collega's end ecollega' s vand e andere onderzoeksgroepen indi t gebouw wil ik bedanken voor de gezellige sfeer envoo r hun hulp. Met de mensenva n deVlinderstichtin g kon ik altijd mijnenthousiasm e voor vlinders delen. Dick en Mathilde, Michiel enJose , Kars enAnnet , Henkjan en Nettie,Gerard , Ingee n Maarten,Wilma , Saskia, Isabel, Henk, Victor,Vivian ,Mariska ,Titi a en Frank, Robert, Liesbeth, Liesbeth,Lia , Jolanda, Els, Poppe, Roelie,d e mensen vand e bieb en iedereen diee r tijdelijk bijwa se n is:bedank t voor de leukesfeer , deondersteunin g end e altijd open deur.Victo r heeft een pluimverdien t voor het makenva nd e mooieomslag ! Delede nva nd ewerkgroe p Evolutiebiologie in Leidenwi l ik bedanken voor de prettige samenwerking, met name Rinny, Helene, Fanja, Peter, Hans,Cock , Klaus en Marjanda. Deafstan d maakted esamenwerkin g niet gemakkelijk, maar bijjulli e was het altijdz o inspirerend,da t ikregelmati g detrei n naar huis hebgemist . Destudenten ,waarme e iksame n mochtwerke n endi evee ldat a voor mij hebben verzameld: Ivo, Marcel,Simon ,Jurgen ,Tjeerd , Natalie,Anne , Menno, Maarten, Mark,Alexander , Jasper, Evelien,Suzann e ye lgrup o espanolAsu n Hidalgo Lopez, Esther Rebon Sartal yJavie r del Rio Diez. Gracias! Hetwa s leuk metjullie ! Enoo k ik hebe r veelva ngeleerd . Paul Kreijger eni nd e beginjaren Ans Raadsen hebbeno pd e monitoring route ind e Moerputten iedere week vanApri l tot Oktober vlinders geteld.Pau l heeft ook nogvel e kilometers perfiet s naar pimpernelblauwtjes gezocht. Zozij n belangrijke gegevens verzameld.

230 Bedankt! Bij hetopzette n vand e experimentenware n mijn krachten vaak niet toereikend. Desterk e mannen, die mij hielpen,ware n IvoRaemakers , Ruudva n Kats, Monte Gardenier en Maarten van Steenis. Ookjulli e bedankt! Demedewerker sva n Staatsbosbeheer, inhe t bijzonder Janny Resoort enJos eVoerma n mogen nietworde nvergeten .O pslecht s eenenke l incident na kan ikteru g kijken opvel ejare n van prettige samenwerking,e n ik hoopda tda t nog langz oza l blijven.Oo kd e samenwerking metall e participanten van het Convenant Pimpernelblauwtje hoop ik nog lange n meteve nvee l plezier als in hetverlede n voort tezetten . De herintroductie end e evaluatie gedurende deeerst ejare n gebeurden inopdrach t van het Ministerie van Landbouw, Natuurbeheer enVisserij .I n die periode heb ik bijzonder prettig samengewerk t metJa nWille m Sneep. Rienk deJon g (Naturalis Leiden) wil ik bedanken omdat hij meall e /Wacu//nea-vlindersui td ecollecti eva n het museumte r beschikking heeft gesteld,zoda t ikoo k een blik naar hetverr e verleden konwerpen .Nic o Kok, Nico en Erika Elfferich en iedereen die mee isgewees t naard e Moerputten,wil l ikdanke n voorjulli e enthousiasme. Dankzijjulli e bleven de pimpernel-blauwtjes ook voor mij heel bijzonder. I would also liket othan k my Polish colleagues Michal Woyciechowski and Edyta Figurny and their families.Than k youfo r your hospitality. You changedth ewor ktrip st o Poland intomarvellou s holidays. Enda t kwam ook doorjulli e begeleiding enassistentie , Mariska enSimon ! Gracias aand e Flamenco-Aficionao's,voora l Ida enAlois ,d e danslessen gaven deenergi e om metalle s andere door tegaan .Ee n dikke bezovoo r Ineke,di e meva nworksho p tot concert tot Penasleepte . Ik heb genoten van alle Sevillanas metjou , envoora l die zondag ind e Westergasfabriek inAmsterda m zal ik nooit vergeten: hetwa sz o mooi toenji j zongterwij l wij midden ind e nacht opd etrei nwachtten . Alle vrienden in Nederland, Duitsland,Amerika , Belgie enwaa r ook ter wereld wil ik bedanken voorjulli e interesse, hulpe ngeduld .D e gesprekken metjulli e waren niet alleen afleiding,maa roo k een bronva n creatieve ideeen. Ik hebvas t zovaa k over "mijn" blauwtjes gepraat dat jullie alien noodgedwongen vlinderexperts zijngeworden . Erkom tee n feest om hetwee r goed te maken! Last, but not least: Marii mferne n Kanada.Wei twe g unddoc h sonah . Inzwischen sind es 30Jahr e Freundschaft, undda s merktfrau ,wen n es darauf ankommt. Danke!

231 Curriculum vitae

IrmaWynhof f werdgebore no p2 5decembe r 1960i n Hasselt (Duitsland). In 1980 behaalde zij haarAbitu raa n hetstedelij k lyceum Johanna-Sebus-Schulete Kleve.Z e begon in hetzelfdejaa r met haar studie biologie aan de Katholieke Universiteit in Nijmegen, Nederland.Da t leiddeerto e datz ezic h definitief inda t landvestigde . In haar studie concentreerde zezic hvolledi g opd eecologi e enstudeerd e af indri e vakken: Plantenecologie envegetatiekunde , Dierecologie en natuurbeheer, enal sderd e vak Aquatische ecologie.Tijden s destudie , dieto t 1988duurde ,wer d het interesse invlinder s gewekt, enlate r heeft zevoornamelij k aan ecologische vraagstellingen omtrentd e levenswijze van deze insectengroep gewerkt. Haar eerste project omvatte het "Beschermingsplan Dagvlinders"voo r het Ministerieva n Landbouw, Natuurbeheer enVisserij . Naast een grote hoeveelheid kleine projecten begon ze in 1989 met devoorbereidin g van de herintroductie vantwe eo p mierenparasiterend e vlindersoorten, het pimpernelblauwtje en het donker pimpernelblauwtje. In 1990werd ,eveneen s ondersteund door het Ministerie van Landbouw, Natuurbeheer enVisseri j de herintroductie uitgevoerd. Irmawer d gegrepen door decomplexitei tva n hetvlinder-mier - systeeme n stortte zicho p deevaluati e vand e herintroductie end e ontrafeling van enkele ecologische problemenva n dezevlinders . Tot 1995 voerde zij haarwerkzaamhede n uital s parttimegastmedewerkste r vand e Vakgroep Terrestrische Oecologie en Natuurbeheer end einmiddel s opgerichte Vlinderstichting. In 1995 begonzij ,naas t haarwer k opd e Vlinderstichting, metee n promotie-project bij Professor Herbert Prinsva n de Leerstoelgroep Natuurbeheer ind eTrope n en Ecologieva n Vertebraten,Wageningen ,e n Professor Paul Brakefield, Instituut voor Ecologische en Evolutionaire Wetenschappen, Leiden. Indi t project werden devoorlopig e conclusies indetai l uitgewerkt engetoetst. Daarnaast werd haard e mogelijkheidgebode n om naast ecologische ook genetische vraagstukken te bewerken. Het resultaat ervan isdi t proefschrift. Tevens heeft zij twee boeken enee n CD-romove r vlinders gepubliceerd.

Irma isgetrouw d met Chris van Swaay. In 1991e n 1993heef t haar leven nieuwe dimensies gekregen door degeboort e van haardochte r Eva en haarzoo n Bosse.Zi j gaat graag inzonnig e streken camperen. Inhaa r vrije tijd luistert ze naar endans tz eo p Flamenco muziek. Indez e muzieksoort heeft zezic h ontwikkelt tot eenecht e "aficionao".

233 Druk:Va n Gils Grafisch Service Centrum,Wageningen .

Foto's buitenkant: KarsVeling , Henkjan Kievit Overige foto's: Henkjan Kievit, Gerard Oostermeijer, Chrisva n Swaay, Jeremy Thomas, KarsVeling , Irma Wynhoff.

Hetonderzoe k is medegefinancier d door het Ministerie van Landbouw, Natuurbeheer enVisserij ,d e Provincie Noord-Brabant, deVlinderstichting , Wageningen Universiteit en Universiteit Leiden.

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