Biodivers Conserv (2008) 17:1429–1437 DOI 10.1007/s10531-008-9350-3

ORIGINAL PAPER

Can the Bronze, Cacyreus marshalli, become a threat for European biodiversity?

Ambra Quacchia · Chiara Ferracini · Simona Bonelli · Emilio Balletto · Alberto Alma

Received: 31 July 2007 / Accepted: 25 January 2008 / Published online: 20 February 2008 © Springer Science+Business Media B.V. 2008

Abstract Cacyreus marshalli Butler is an invasive species in many parts of Europe and Mediterranean area. In Europe, its larvae normally feed on pelargoniums. We investigated its potential to spread to native Geranium spp. and evaluated the conservation risks that such a shift would pose for both native and cohabitant butterXies. The host plant preferences of the were investigated under controlled conditions. Studies included both no-choice and multi-choice tests, respectively using 9 and 6 Italian native Geranium spp. Host plant preferences were evaluated by counting the number of eggs laid on individual plants and following butterXy development until adult emergence. Under no- choice conditions, at least one egg was recorded on each tested plant, except for G. phaeum L. All the plants on which oviposition occurred were fully suitable for larval development. The butterXy, however, clearly preferred three species, i.e. G. pratense L., G. sanguineum L. and G. sylvaticum L. for oviposition. In multi-choice trials, females laid at least one egg on all the tested plants, with a preference for G. pratense and G. sylvaticum. In presence of Pelargonium spp. plants, however, no oviposition was observed on any Geranium spp. We assessed oVspring Wtness measuring their wingspan. No statistical diVerences were detected in the wingspan between adults emerged from Geranium and Pelargonium. Cacyreus mar- shalli represents a potential threat for both native geraniums and for Geranium-consuming lycaenids, such as nicias Meigen and eumedon Esper.

Keywords Host plant preference · Geranium · Pelargonium · Biodiversity conservation · Competition with native species

A. Quacchia · C. Ferracini · A. Alma (&) Di.Va.P.R.A. – Entomologia e Zoologia applicate all’Ambiente “Carlo Vidano”, University of Torino, via Leonardo da Vinci, 44, 10095 Grugliasco, Torino, Italy e-mail: [email protected]

S. Bonelli · E. Balletto Dipartimento di Biologia Animale e dell’Uomo, University of Torino, via Accademia Albertina 13, 10123 Torino, Italy 1 C 1430 Biodivers Conserv (2008) 17:1429–1437

Introduction

Cacyreus marshalli Butler (: ) is a small lycaenid butterXy, recently introduced into Europe, where its larvae attack all ornamental Pelargonium species. Its region of origin is in South Africa, where it was recorded from the Cape Provinces, Natal, Lesotho, Swaziland Botswana, Zimbabwe and Mozambique (Clark and Dickson 1971; OEPP 2002). In Europe, a viable colony was Wrst observed in Mallorca (Sarto i Monteys 1991). During the past 15 years, C. marshalli has colonized much of southern Europe, where it has become established (Sarto i Monteys 1992). In Italy it was Wrst observed in Rome in 1996 (Trematerra et al. 1997) and quickly spread to most other regions. The spread occurred initially mainly along the Tyrrhenian and the Adriatic costal areas, but extended inlands very soon (Favilli and Manganelli 2006). In South Africa, the main larval host plants are Pelargonium spp. and Geranium spp. (Clark and Dickson 1971). Eggs are laid near the Xower buds or less frequently on the leaves. Caterpillars are found within the Xower buds or inside the stem, where they bore through. Entrance holes in buds and stems are easy to detect. Once attacked, the stems turn blackish. The species is polyvoltine, prob- ably producing up to six generations per year. Its Xight period extends well into September or October (Favilli and Manganelli 2006). Problems in controlling C. marshalli are not very important in nurseries, where regular precautionary insecticide sprays may be carried out, but rather when plants reach the con- sumer. As already pointed out by Sarto i Monteys (1992), in fact, contact insecticides have no eVect, due to the endophytic habits of the larva. As a consequence of the fast spread and dangerousness of this pest, pelargoniums were included among the species requiring quar- antine and C. marshalli was listed as an A2 quarantine pest by the European and Mediterra- nean Plant Protection Organization (OEPP 2002). Since many cranesbills species (Geranium) are included in the Italian Xora and due to the importance of preserving the richness and diversity of our natural heritage, we carried out a study to establish the possible adaptation of C. marshalli to native Geranium species. The potential competition between the Geranium Bronze and other native geranium-con- suming lycaenids, such as Esper and Meigen, is also dis- cussed. Both of these species are currently supposed to be not-threatened and are not listed in the Habitats Directive annexes.

Materials and methods

Monitoring

A weekly monitoring programme, having the aim to detect the presence of C. marshalli, was carried out from June to September over a 2-year period, between 2005 and 2006, in many parts of Piedmont and of the Aosta Valley. All sites occurred in built-up areas, homo- geneously dispersed across the region. Ornamental Pelargonium spp. plants were visually checked and the presence/absence of the lycaenid was recorded.

Field collection and laboratory rearing

The larvae, pupae and adults of the Geranium Bronze were collected in the Weld from ornamental Pelargonium zonale L. (Aiton) and P. peltatum L. (Aiton) plants, and used to set up a mass rearing stock, as well as to obtain the necessary supply of individuals for our 1 C Biodivers Conserv (2008) 17:1429–1437 1431 experiments. Larvae were maintained on Xowered Pelargonium potted plants. Both the mass rearing and the experimental trials were conducted in cages (1 £ 1 £ 1 m) having a stainless steel frame structure supporting a plastic -proof net (mesh 0.23 £ 0.23 mm). Host plant preferences were investigated on a number of native Geranium species, which commonly occur in many mountainous and hilly habitats of northwestern Italy (Table 1). Leaf areas were roughly equivalent among plants. Since the butterXy clearly pre- ferred to oviposit on Xower buds and due to diYculties in having all the plants Xowering at the same time, all Xower buds were removed. We did not investigate the possible inXuence of the plants’ phenological traits. A small piece of cotton absorbed with a diluted honey solution was placed on a slide in the middle of the cage, in addition to one Calendula spp. Xowered potted plant, to supply feeding to the adults. We provided daily irrigation, to maintain the growth and health of the plants. The potential host range of C. marshalli was investigated by conducting both no-choice and multi-choice oviposition tests on various Geranium spp. plants.

No-choice experiments

For these experiments we used nine potential larval host-species (Geranium dissectum L., G. lucidum L., G. molle L., G. phaeum L., G. pratense L., G. pyrenaicum L., G. robertianum L., G. sanguineum L., G. sylvaticum L.), and P. peltatum. We performed no-choice preference tests by placing four potted plants of the same species in a cage where we introduced also 20 newly emerged adults.

Multi-choice experiments

We used six potential larval host-species (G. nodosum L., G. phaeum, G. pratense, G. pyre- naicum, G. sanguineum, G. sylvaticum). Six potted plants, each of a diVerent species were simultaneously presented to 30 adults, in various combinations. Finally, we performed an additional multi-choice test, as previously described, by add- ing to the six Geranium spp. plants also two P. peltatum potted plants.

Trials

Five replicates were conducted for all the trials. We checked all plants daily, to detect ovipositions. After all the butterXies were dead, we removed all the dead adults and sexed them under a binocular lens. We moved each plant to a separate plexiglas cage (200 £ 200 £ 300 mm). We evaluated oviposition preferences by counting the number of eggs laid on each tested plant. We checked oVspring development daily. In case of high oviposition, a maximum of three eggs per leaf was kept. The other eggs were then removed with a small brush, to avoid possible larval cannibalism. The adults emerged from the eggs laid on each plant species were transferred to cages containing P. zonale potted plants, to test if they were able to mate and oviposit. Observations were made daily. Again, once the adults were dead, we sexed them and measured their wingspan under a binocular lens (15£ magniWcation) equipped with an ocular micrometer. All the adults used in the trials were less than 24 h old and all the tests were performed in the heated glasshouse of the Agronomy Faculty, where temperatures ranged from 18–20

1 C 1432 Biodivers Conserv (2008) 17:1429–1437 Italian distribution 0–1,600 0–1,200 0–2,300 200–1,900 (a.s.l.) and edges wood wood edges and rocks and hedge walls, banks, places androadsides waste and xelophilous bushes andmountain rock ledges Meadowsroadsides and Pastures, barren uncultivateds 500–1,300 Moist shady positionsin undergrowth, Coppices, edges wood, undergrowth Meadows,hedgebanks, damp woods a a,b a,b a,b a,b ) pratense pyrenaicum robertianum sanguineum sylvaticum 2002 Geranium Geranium Geranium Geranium Geranium Species Habitat Altitude m Italian distribution 0–1,300 0–1,400 0–1,000 (a.s.l.) spp. used in the trials (from Pignatti Pignatti (from trials the in used spp. Geranium grassland and hedgebanks hedgebanks and grassland hedgebanks, on calcareous soils places and cultivated ground Cultivated and waste ground, walls and Shady rocks, Dry grassland, dunes, waste Broad-leaf wood Meadows and clearings wood 100–1,300 500–2,000 a b a a,b Environmental features of the a molle lucidum nodosum phaeum dissectum Plants used in the multi-choice tests multi-choice in the used Plants Plants used inthe no-choice tests Geranium Geranium Species Habitat Altitude m Table 1 a b Geranium Geranium Geranium Geranium Geranium Geranium 1 C Biodivers Conserv (2008) 17:1429–1437 1433

(night) to 30–35°C (day) and the relative humidity from 50 to 90%, with a 15L:9D photo- period.

Statistics

DiVerences in eggs numbers were tested with a non parametric ANOVA test (Kruskal– Wallis) with a Dunnett’s T3 correction of signiWcance levels. DiVerences in wingspan between hosts and sexes were tested using Dunnett’s t test. All data were handled by SPSS 13.0 and signiWcance levels were taken to  = 0.001 in all tests.

Results

In the distribution area of northwestern Italy, a well-established C. marshalli populations was found, in all the monitored sites (Fig. 1). Zonal and ivy-leafed pelargoniums were always present in the vicinity. Although this map includes a number of unpublished data, the real distribution is certainly much denser than shown here, since many observations remain unreported. The oviposition rates observed in the host choice tests are shown in Table 2. Eggs were laid both on the upper and on the lower leaf surfaces, less frequently on stems. We recorded the Wrst eggs from 3 to 9 days after the adults were released into the cage. The adult longev- ity was on average of 8.25 § 1.15 days and it took about 26.92 § 2.14 days to obtain the adults once the eggs were laid.

No-choice experiments

Under no-choice conditions, females oviposited on all the tested plants, except on G. phaeum. The greater number of eggs was laid on G. sanguineum, G. pratense and G. sylvaticum, with 189, 380 and 384 eggs, respectively (cumulative data). A statistically signiWcant diVerence was demonstrated to exist between females ovipositing on Geranium and on Pel- argonium (Table 2).

Multi-choice experiments

Under multi-choice conditions, females laid eggs on all the tested plant species. Fewer eggs were found on G. sanguineum and on G. pyrenaicum, while the greater oviposition rates were observed on G. sylvaticum and G. pratense, with 86 and 91 eggs, respectively. These data were signiWcantly diVerent from those observed on the other species. When adults were oVered a choice between Geranium and Pelargonium plants, they only chose the latter (Table 2). Except for those laid on G. nodosum and G. phaeum under multi-choice conditions, all the other eggs completed their development. The trophic activity of the larvae proved fatal for the plants, which withered in some weeks. The wingspan of the oVspring was in all the cases included in the range reported in liter- ature (#: 15–23 mm; $: 18–27 mm) (OEPP 2002). No statistically signiWcant diVerences were found between the individuals emerged from Geranium spp. and Pelargonium spp., both for females and males (Table 3). Once transferred into the cages with P. zonale potted plants, all the adults emerged from the eggs laid on Geranium were able to mate and ovipo- sition occurred. The sex ratio of Geranium-reared individuals was of 1.24 ##/$$.

1 C 1434 Biodivers Conserv (2008) 17:1429–1437

Fig. 1 The known Italian distribution of Cacyreus marshalli Butler (updated from Balletto et al. 2007)

Discussion

The heavy commercial trade, both by air and land, with other countries, combined to the ever-increasing tourist traYc, is favouring the incidental introduction of phytophagous into Italy (Pellizzari et al. 2005). Cacyreus marshalli is just one of the many inva- sive pest species which recently reached Italy, initially because of the widespread exchange 1 C Biodivers Conserv (2008) 17:1429–1437 1435

Table 2 Oviposition rate (replicates combined) on Cacyreus marshalli Butler on the tested plants

Host species No. of eggs laid in No. of eggs laid in No. of eggs laid in the no-choice test the multi-choice testa the multi-choice testb

Geranium dissectum 7a – – Geranium lucidum 1b – – Geranium molle 17c – Geranium nodosum –6a0a Geranium phaeum 0b 8a 0a Geranium pratense 380d 91ab 0a Geranium pyrenaicum 3b 2a 0a Geranium robertianum 47e – – Geranium sanguineum 189f 1a 0a Geranium sylvaticum 384d 86b 0a Pelargonium peltatum 3843g – 1877b a Multi-choice test without Pelargonium peltatum plants b Multi-choice test with Pelargonium peltatum plants Values in the columns followed by the same letter do not diVer signiWcantly by Dunnett t test (P < 0.001)

Table 3 Number and wingspan Host species No. of adults Wingspan (§SE) of progeny of Cacyreus examined marshalli Butler #$# $

Geranium dissectum 5 4 19.08 § 0.46a 21.50 § 0.86a Geranium lucidum 1 0 17.88 § 0.00 – Geranium molle 6 4 18.58 § 0.24a 23.26 § 0.37a Geranium pratense 5 1 19.73 § 0.67a 22.18 § 0.00 Geranium pyrenaicum 2 1 20.85 § 0.51a 24.14 § 0.00 Geranium robertianum 7 9 19.49 § 0.32a 22.35 § 0.32a Values in the columns followed Geranium sanguineum 14 11 20.36 § 0.27a 23.88 § 0.26a V by the same letter do not di er Geranium sylvaticum 11 8 19.64 § 0.32a 23.68 § 0.26a W signi cantly by Dunnett t test Pelargonium peltatum 8 9 19.63 § 0.41a 22.49 § 0.64a (P < 0.001) of plants, especially pelargoniums, with some important producers and exporters such as Spain, Germany, the Netherlands etc. Once introduced, also the rapid spread of this species to most Italian regions is often ascribed to passive human transportation from site to site, together with pelargonium plants or shoots. How much of this spread can be ascribed to active dispersal, however, remains unclear, since this species vagility have never been investigated to some detail. This study has demonstrated that oviposition can occur easily on many Italian native Geranium species. Since some wild-growing geraniums are host plants of C. marshalli in South Africa, this may be seen as a partially expected Wnding, even though European spe- cies are certainly quite diVerent from those occurring in South Africa. Both in no-choice and in multi-choice trials, we have observed that females clearly prefer G. pratense, G. san- guineum and G. sylvaticum plants, while the least desirable are G. nodosum and G. phaeum. However, even if the number of eggs recorded on some species was of over 100 per plant, when Geranium spp. and Pelargonium spp. were oVered together, all females oviposited only on the latter. Some similar, although somewhat anecdotal, tests carried out by Sarto i Monteys (1994) on G. pyrenaicum, G. robertianum and G. rotundifolium L. gave similar results. Tiberghien 1 C 1436 Biodivers Conserv (2008) 17:1429–1437 and Vesco (2003), in contrast, have not obtained any oviposition on Geranium spp. and reported that larvae initially reared on Pelargonium and later transferred on Geranium plants starved to death. The latter authors, however, have provided no information about the species or the number of plants tested. In our case, only the eggs laid on G. nodosum and G. phaeum during the multi-choice experiments have failed to develop and no eggs have been observed on these species even under no-choice conditions. Cacyreus marshalli has never been reported as a pest species in its area of origin, proba- bly because some autochthonous parasitoids and predators are able to keep its population under the damage threshold. In our study we have not observed any parasitoid or predator. Among parasitoids, only Sarto i Monteys reports one egg of C. marshalli parasitized by Trichogramma evanescens Westwood (Favilli and Manganelli 2006). The introduction into Europe of the Geranium Bronze is having great impact on the nursery sector, with a consequent decrease in the demand of Pelargonium, which are ever more often replaced by customers with other ornamental plants. Even though in nurseries the control of the pest may be carried out through the aid of common insecticides, this is not feasible elsewhere, particularly in the mountainous and hilly habitats where wild Geranium species commonly occur. For the regions of Piedmont and Aosta Valley, Pignatti (2002) reports the occurrence of 18 Geranium species. Should an adaptation to these native species ever occur, C. marshalli would become a real threat also for the native Xora, as well as for local biodiversity. A fur- ther threat potentially posed by this species, in fact, is in the possible competition with some indigenous lycaenid species, such as E. eumedon and A. nicias. These species, in fact, can only feed on Geranium, notably on G. sylvaticum and G. pratense, which are the most easily accepted species by C. marshalli females for oviposition. Since the Geranium Bronze is multivoltine and accepts to lay on leaves and stems and not only on Xowers, it would probably out-compete the other two, both monovoltine, in areas of sympatry. Of par- ticular interest, in this respect, is our recent Wnding of C. marshalli at Courmayeur (prov- ince of Aosta, 1,224 m), less than 1 km away from the nearest known population of E. eumedon and at less than 10 km from the nearest A. nicias (Balletto et al. 2007); there is little doubt that such distances can be passed by this species. As concerns the probability that a shift onto the native Geranium species can occur in C. marshalli, the voltinism-suit- ability hypothesis suggests that the diet breadth is restricted to hosts supporting the most rapid larval development where populations are near the thermal limits. In contrast when the same number of generation can be achieved without diYculty, relaxed selection will permit females to oviposit on hosts on which larval growth is lower (Scriber and Leder- house 1992). Variation in oviposition preference behaviour among diVerent host species is heritable and responsive to selection (Thompson and Pellmyr 1991; Renwick and Chew 1994). The evolutionary mechanisms allowing or favouring the shift to a “new” larval host plant are rather obscure. Ladner and Altizer (2005), however, have argued that either underlying genetic variation or maternal eVects can be involved in this process. To partially elucidate the mechanism governing this trait, it will be interesting to investigate if the adult C. marshalli females emerging from larvae that developed on native Geranium species are more likely to lay their eggs on the same or on a similar plant species, or if they will continue to prefer Pelargonium plants, in multiple choice experiments. Some authors have argued that species invasions can increase, rather than reduce, species diversity (see Hulme 2003 for a detailed analysis). This may be true also for C. marshalli, but only in man-dominated, urban ecosystems. Should this species be able to spread to natural environments it would probably cause considerable ecosystem impact, by 1 C Biodivers Conserv (2008) 17:1429–1437 1437 aVecting both native geraniums and geranium eating lycaenids. Even apart from this, as suggested by Trematerra and Parenzan (2003), the adaptation of this lycaenid to autochtho- nous plants may favour its spread and establishment not only in Italy and in Mediterranean areas, but also deep into mainland continental Europe, causing serious economic and environmental losses.

Acknowledgments This research was part of the Interreg III-ALCOTRA project “Strategie di lotta biolog- ica per contenere Wtofagi esotici di recente introduzione nella zona transfrontaliera italo-francese, a salvaguar- dia delle diverse realtà ambientali” and funded by Regione Piemonte and Regione Valle d’Aosta. Finally, we wish to thank an anonymous referee for his many useful comments.

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